http://2009.igem.org/wiki/index.php?title=Special:Contributions/Aurel&feed=atom&limit=50&target=Aurel&year=&month=2009.igem.org - User contributions [en]2024-03-29T12:42:41ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-22T01:50:05Z<p>Aurel: /* Results [1,2] */</p>
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= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the ability to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
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<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction which has been extracted from a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offers a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the adenovirus 5[[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its prefix and suffix. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
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<br />
First and second pair for genes extraction: <br><br />
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D protein of the Lambda phage: <br><br />
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Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
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Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
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Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
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Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure 1. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 11) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
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A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure 2 shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
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Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure 2). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragment coding for RGD sequence alone has a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Conclusion ==<br />
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To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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= Antitumoral Plasmide =<br />
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== Context ==<br />
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In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
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The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing into tumor cells the wild type genes coding for functional tumor suppressors.<br><br />
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The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] allowed us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
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[[image: gènes mutés eng.jpeg|center]]<br />
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These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== The objective ==<br />
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The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
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== Experimental approach ==<br />
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=== Cancer cell line and reported gene ===<br />
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Cancer cell lines, which have a mutation of tumor suppressor gene has been selected from our database. We also possess a wild type version of the TP53 gene. Then we choose the prostatic cancer p53 mutated DU-145 in the goals to test if bringing the wild type version of the p53 protein (p53wt) in the DU-145 cell line allows to induce the apoptotic process. <br><br />
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<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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=== TP53 gene incorporation ===<br />
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Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
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<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
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<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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=== Apoptosis detection ===<br />
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Detection of apoptotic cells is done by the annexin V assay: <br><br />
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In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
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<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
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Work as much as possible in the dark (fluorophores are photolabile) <br><br />
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<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== The running of the study ==<br />
<br />
To analyse the timing of plasmid expression in the DU-145 cell line, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48 hours after the plasmid electroporation. By coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. We use a control without the plasmid pcDNA3 CMV+p53wt to quatify the level of death cells causes by the electroporation and by the culture transfert. <br><br />
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Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
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Here is the allocation planning of electroporations: <br><br />
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[[image:planning eng.jpeg|center]] <br />
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Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9 a.m, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 4 p.m, day 3). <br><br />
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The first cytometric analyze allowed us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, allowed us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
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By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Results [1,2] ==<br />
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Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell population during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analysis. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
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Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let us to try a second experiment. <br><br />
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However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
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Infected by AdCMV.p53, cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin by the genome fragmentation observation, consequence to the CAD (Caspase Activated DNase) activity, a specific endonuclease of the apoptotic process. <br><br />
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A MTT test allowed to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
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In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells were dead (figure 3). <br><br />
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<u><i>According to this study, it appears clearly that to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decreases significantly the tumor population.</i></u><br><br />
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Similar results were reported in the study leaded by Corrado Cirielli (in 1999) by using similar analyses on a U251 cancer strain from a glioma. <br><br />
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<dt> Morphologic analysis of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
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Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the test population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
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<dt> AdCMV.p53 effect on DNA fragmentation :<br><br />
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<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
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After AdCMV.p53 infection, U-251 cells show a fragmentation of their genome, characteristic of the apoptosis process (laddering pattern).<br><br />
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<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
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<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
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<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
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<dd><u><i>This study shows one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Concept#drapeau|DVS]], application in the fight against non small cell lung cancer, is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Treatement_modelingTeam:SupBiotech-Paris/Treatement modeling2009-10-22T01:38:17Z<p>Aurel: /* First step : Tumour development according to time */</p>
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= Modelling of DVS efficiency on a lung tumour =<br />
<br />
== Context ==<br />
<br />
Non-small cell lung carcinoma, or NSCLC, is an aggressive cancer, with a relatively high speed growth. Treatments are often ineficient, because the tumour growth is faster than the elimination by the drug.<br><br />
<br />
== Objective ==<br />
<br />
We have decided to model our treatment efficacy for this kind of tumour. Therefore we have modelled the tumour progression, our treatment evolution and efficacy.<br />
The objective of the modelling is to verify if our treatment is able to eliminate the entire tumour.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
== Model segmentation ==<br />
<br />
First, we had to recreate [[Team:SupBiotech-Paris/Concept#DVS|DVS]] complete mechanism and the tumour evolution. Then, for each step of the treatment, we have identified all the paramters that intervene, their actions and their interactions, in order to determine the model equations.<br><br />
<br />
To simplifly the equation we have devided the mechanism and we have modelled each step separately.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
=== Tumor and DVS evolution versus time ===<br />
<br />
==== First step : Tumour development according to time ====<br />
<br />
We consider the '''tumour is non metastatic and its growth is exponential'''.<br><br />
Let the tumour have a volume V1 in cm3 at an instant t1.<br><br />
Let the same tumour, at an instant t2, have a volume V2.<br><br />
The tumour is considered in exponetial growth phase and without metastasis therefore its development equation, '''Tumor Growth Rate (TGR)''', is equal to :<br><br />
[[Image :TGR.jpg|center|200px]] <br />
Thus, the '''tumour volume according to the time (V(t))''' is equal to :<br><br />
[[Image :V(t).jpg|center|300px]] <br />
Finally, knowing the ''' Average volume of a cancerous cell (Vcc)''' (experimental data), if we regard the tumour as fraught (without cavity or blood vessel), we can determine that the '''Number of cancerous cells according to time (Nc(t))''', without treatment effect, is equal to :<br><br />
[[Image :N(c).jpg|center|150px]] <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Second step : Development of the tissue vector====<br />
<br />
The [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] is injected to the patient at an instant t, near t2. The '''Number of injected vectors (Nbi)''' is 1x10^6. The pulmonary tropism of the vector isn’t perfect, only a '''Percentage (Pp)''' goes to the lung. The total number of [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] in the body increases, because this vector is bacterial and therefore possesses a '''Doubling period (DTB)'''.<br><br />
Thus we can establish that the '''[[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] number in the lungs (Nb(t))''' is equal to:<br><br />
[[Image :Nb(t).jpg|center|330px]] <br />
<br />
The number of [[Team:SupBiotech-Paris/Concept1#drapeau|Tissue vectors]] increases until injection of doxycycline. F rom then, tissue vectors lysis releases the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] in the lung.<br><br />
<br />
This injection time is not insignificant. Indeed, si if we wait long enough, [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]] number is sufficient to eliminate the tumour or at least to significantly reduce it. On the other hand, if we wait too long, a higher dose of doxycycline (and so potentially toxic) is necessary for [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] release.<br><br />
<br />
Thus we can use modelling to determine the ''' optimal injection time of doxycyline (Tdox)'''.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Third step : Release of the cell vector ====<br />
<br />
Once the doxycycline injected, the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] is released. The [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number is proportional to the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]] number in the lung. And yet, we know the average value of '''recombinant phage vectors released by M. ''avium'' (Npl)''' is equal to 100.<br><br />
We can write '''[[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number at the injection instant (Np(Tdox))''' is equal to :<br><br />
[[Image :Np(t)1.jpg|center|200px]]<br />
<br />
The [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number does not increase such as the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]]. Indeed, it decreases with time, because of the phage vector stability and of its cell penetration (to release the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmide]]).<br><br />
Its stability in the blood is equal to the '''phage vector deterioration constant (kdeg)''' according to time. If we add this constant to the '''[[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number equation according to time (Np(t))''' we obtain the following formula :<br><br />
[[Image :Np(t)2.jpg|center|300px]] <br />
<br />
The phage vector dispersion steps in the tumour and for cell penetration are the steps below '''Fourth''' and '''Fifth''') because of their complexity.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
=== DVS Efficiency ===<br />
<br />
So, we determined:<br />
:* The size of the tumor versus time (initial volume + growth)<br />
:* The amount of tissue vector versus time<br />
:* The amount of released [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] for a tissue vector <br />
<br />
Now, we're going to determine the efficiency of our vectors for penetring cancer cells. <br><br />
For that we are studying:<br />
:* The area of dispersal [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]]<br />
:* The importance of the cellular internalization of the vector in cancer cells.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Fourth step : The cell vector dispersion ====<br />
<br />
Here, we are looking for determine the maximum area that a phage can cover.<br />
This requires knowing:<br />
<br />
:* The spread of phages in the bloodstream<br />
:* Their diffusion through the walls of blood vessels<br />
:* The surface of a cancer cell<br />
<br />
For our modeling, we consider the blood as a '''Newtonian fluid with a constant velocity Vmax'''. Here, We neglect the heart-related jolts and turbulent flows caused by the cavities of the blood epithelium.<br><br />
<br />
The [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] moves along two axes. An X axis in the direction of blood flow and a Y axis orthogonal to the axis X.<br><br />
<br />
[[Image:RepèremouvementmécaniqueEn.png|center|400px]]<br><br />
<br />
=====The phage propagation in the bloodstream=====<br />
<br />
The movement '''in X''' depends solely on the '''propagation of phages''' the vessel due to blood flow. Indeed, we neglect the diffusion which takes place also along the X axis because it is 1000 times less than the propagation of particles in the blood (due to the importance of blood flow). The cellular carriers are moving at '''speeds spread on a dish''' from, Vmax in the center of the vessel at V0 against the vessel wall.<br><br />
<br />
[[Image:répartitionvitesseparaboliqueEN.png|center|500px]]<br><br />
<br />
The speed of phages decrease in approaching the vessel walls due to the friction forces which are opposing to the movement.<br><br />
<br />
We can determine how long the particle (with a Vmax velocity), ie the particles in the center of the ship, reached the end. This gives the time necessary to internalize all the phages of the bacteria.<br><br />
<br />
=====The diffusion through the walls of blood vessels=====<br />
<br />
The movement '''in Y''' is the distribution of phages in the blood (j(n)). It depends on the equation of diffusion of a particle (n) in a fluid (Fick's Law).<br><br />
<br />
[[Image:Equation diffusion du phage.png|center|170px]]<br><br />
<br />
With n the number of particles (phages), grad n the difference between the concentrations and D the diffusion coefficient.<br />
The cellular distribution of vectors within the blood vessel and then through the wall is a phenomenon of diffusion with output. So, there will always be a strong gradient of concentration of phage in the blood. We can therefore say that the gradient is constant (equal to 1) over time. Thus the diffusion rate (j(n)) is equal to D.<br />
<br />
<br />
=====Dispersal area of phage=====<br />
<br />
When we combine moving '''Y''' ('''diffusion rate''') and moving '''in X''' ('''blood flow velocity'''), we obtain, after integration on the '''perimeter of a blood vessel''', the action surface of [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]]. Then, we are able to determine the number of cancer cells per 100 [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] destroyed or 1 [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]].<br />
<br />
The diffusion rate of the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] is equal to 0.5 μm.s-1 and the size of a capillary blood is 10μm in diameter. The particle farthest places so 10s to reach the vessel wall.<br />
<br />
With this '''dissemination length''' (10s), the '''blood flow velocity''' (1x10 ^ 3μm.s-1) in the capillaries, and the surface of one cancer cell (1 micron square), we can determine:<br />
<br />
:* The '''length (L)''' covered by the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] released by one [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]].<br />
:* The '''surface (S)''' occupied by phages in blood vessel diameter of 2r.<br />
:* The amount of cancer cells available.<br />
<br />
L = 1 x 10^4 µm<br />
2r = 10 µm<br />
S = 2 x π x L r = 31.4 x 10^4 µm² <br />
<br />
Thus, a [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] can potentially target more than 31 000 cancer cells, yet it has that 100 [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]]. We can make a simplification to say that 100 [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] destroy 100 cancer cells. The efficiency of the dispersion is complete.<br />
<br />
For phage, once reached the wall, comes in the cellular internalization. This model responds to two courses of action.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Step Five: The cell vector internalization ====<br />
<br />
Once in contact with the cell, the cell vector has two possible ways of action.<br />
<br />
:* The vector fix then it detaches from the cell.<br />
:* The vector fix then it is internalized within the cell. <br />
<br />
We can model this according time and the association constant (kon) and dissociation constant (koff) and internalization constant(Kint).<br />
<br />
This gives:<br />
[[Image : EqInt.jpg|center|280px]] <br><br />
<br />
With '''kon''' = 5.10^3 M-1s-1, '''koff''' = 8.10^-3 s-1 and '''Kint''' = 5,78.10^-4 s-1. If we calculate the '''global constant K'''', such as IDP = K' xt, we obtain '''K''''= 361.5 s-1. Thus more than 360 phages are internalized per second in contact with a wall.<br />
<br />
If we compare the time required to internalize a phage compared to waiting times before the cell enters apoptosis in response to the entrance of a vector cell (1 hour), it appears logical to neglect the internalization of cell vectors (IDP = constant = 360 phages/s) in the final equation. <br />
<br />
Thus, with a total efficiency of the phage diffusion, and a neglected internalization time, we can say that the efficiency constant '''λ is equal to 1'''. <br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
== Simultaneous evolution of DVS and tumor ==<br />
<br />
The evolution equation of our model over time is equal to:<br><br />
[[Image :EqFinaleEN.jpg|center|700px]][[Image:Bibou3.png|float|right|150px]]<br />
With :<br><br />
<div style="margin-left: 100px;"><br />
- Nc (t), the number of cancer cells depending time,<br><br />
- V (t), tumor volume,<br><br />
- V1 and V2, two tumor volumes respectively times t1 and t2, <br><br />
- Vcc, the volume of a cancer cell,<br><br />
- Nbi, the number of injected [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]],<br><br />
- Pp, the lung percentage of [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]] relative to the injected dose,<br><br />
- DTB, the doubling time of [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]],<br><br />
- tinj, injection time of the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors],<br><br />
- Npl, the number of [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] released by [[Team:SupBiotech-Paris/Concept1Fr#drapeau|bacteria]].<br><br />
</div><br />
We can neglected (differences between the time or space scales) some factors: <br><br />
<div style="margin-left: 100px;"><br />
- Kdeg, the degradation constant of the phage, because all the phages were internalized in 10s.<br><br />
</div><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
== Treatment simulation ==<br />
<br />
The DVS need two injection:<br />
:* Injection of DVS,<br />
:* Injection of the activator, doxycycline.<br />
Here's a simulator, which calculates how long must we inject doxycycline to completely eliminate the tumor.<br><br />
<html><br />
<center><br />
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src="https://static.igem.org/mediawiki/2009/e/ec/SimulationSBP.swf" <br />
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allowfullscreen="true"<br />
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<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
.<br />
<br />
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<div style="float: right; margin-right: -85px;"><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Treatement_modelingTeam:SupBiotech-Paris/Treatement modeling2009-10-22T01:36:46Z<p>Aurel: /* First step : Tumour development according to time */</p>
<hr />
<div>{{Template:Supbiotechcss13.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Modelling of DVS efficiency on a lung tumour =<br />
<br />
== Context ==<br />
<br />
Non-small cell lung carcinoma, or NSCLC, is an aggressive cancer, with a relatively high speed growth. Treatments are often ineficient, because the tumour growth is faster than the elimination by the drug.<br><br />
<br />
== Objective ==<br />
<br />
We have decided to model our treatment efficacy for this kind of tumour. Therefore we have modelled the tumour progression, our treatment evolution and efficacy.<br />
The objective of the modelling is to verify if our treatment is able to eliminate the entire tumour.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
== Model segmentation ==<br />
<br />
First, we had to recreate [[Team:SupBiotech-Paris/Concept#DVS|DVS]] complete mechanism and the tumour evolution. Then, for each step of the treatment, we have identified all the paramters that intervene, their actions and their interactions, in order to determine the model equations.<br><br />
<br />
To simplifly the equation we have devided the mechanism and we have modelled each step separately.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
=== Tumor and DVS evolution versus time ===<br />
<br />
==== First step : Tumour development according to time ====<br />
<br />
We consider the '''tumour is non metastatic and its growth is exponential'''.<br><br />
Let the tumour have a volume V1 in cm3 at an instant t1.<br><br />
Let the same tumour, at an instant t2, have a volume V2.<br><br />
The tumour is considered in exponetial growth phase and without metastasis therefore its development equation, '''Tumor Growth Rate (TGR)''', is equal to :<br><br />
[[Image :TGR.jpg|center|200px]] <br />
Thus, the'''tumour volume according to the time (V(t))''' is equal to :<br><br />
[[Image :V(t).jpg|center|300px]] <br />
Finally, knowing the ''' Average volume of a cancerous cell (Vcc)''' (experimental data), if we regard the tumour as fraught (without cavity or blood vessel), we can determine that the '''Number of cancerous cells according to time (Nc(t))''', without treatment effect, is equal to :<br><br />
[[Image :N(c).jpg|center|150px]] <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Second step : Development of the tissue vector====<br />
<br />
The [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] is injected to the patient at an instant t, near t2. The '''Number of injected vectors (Nbi)''' is 1x10^6. The pulmonary tropism of the vector isn’t perfect, only a '''Percentage (Pp)''' goes to the lung. The total number of [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] in the body increases, because this vector is bacterial and therefore possesses a '''Doubling period (DTB)'''.<br><br />
Thus we can establish that the '''[[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] number in the lungs (Nb(t))''' is equal to:<br><br />
[[Image :Nb(t).jpg|center|330px]] <br />
<br />
The number of [[Team:SupBiotech-Paris/Concept1#drapeau|Tissue vectors]] increases until injection of doxycycline. F rom then, tissue vectors lysis releases the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] in the lung.<br><br />
<br />
This injection time is not insignificant. Indeed, si if we wait long enough, [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]] number is sufficient to eliminate the tumour or at least to significantly reduce it. On the other hand, if we wait too long, a higher dose of doxycycline (and so potentially toxic) is necessary for [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] release.<br><br />
<br />
Thus we can use modelling to determine the ''' optimal injection time of doxycyline (Tdox)'''.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Third step : Release of the cell vector ====<br />
<br />
Once the doxycycline injected, the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] is released. The [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number is proportional to the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]] number in the lung. And yet, we know the average value of '''recombinant phage vectors released by M. ''avium'' (Npl)''' is equal to 100.<br><br />
We can write '''[[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number at the injection instant (Np(Tdox))''' is equal to :<br><br />
[[Image :Np(t)1.jpg|center|200px]]<br />
<br />
The [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number does not increase such as the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]]. Indeed, it decreases with time, because of the phage vector stability and of its cell penetration (to release the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmide]]).<br><br />
Its stability in the blood is equal to the '''phage vector deterioration constant (kdeg)''' according to time. If we add this constant to the '''[[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] number equation according to time (Np(t))''' we obtain the following formula :<br><br />
[[Image :Np(t)2.jpg|center|300px]] <br />
<br />
The phage vector dispersion steps in the tumour and for cell penetration are the steps below '''Fourth''' and '''Fifth''') because of their complexity.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
=== DVS Efficiency ===<br />
<br />
So, we determined:<br />
:* The size of the tumor versus time (initial volume + growth)<br />
:* The amount of tissue vector versus time<br />
:* The amount of released [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] for a tissue vector <br />
<br />
Now, we're going to determine the efficiency of our vectors for penetring cancer cells. <br><br />
For that we are studying:<br />
:* The area of dispersal [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]]<br />
:* The importance of the cellular internalization of the vector in cancer cells.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Fourth step : The cell vector dispersion ====<br />
<br />
Here, we are looking for determine the maximum area that a phage can cover.<br />
This requires knowing:<br />
<br />
:* The spread of phages in the bloodstream<br />
:* Their diffusion through the walls of blood vessels<br />
:* The surface of a cancer cell<br />
<br />
For our modeling, we consider the blood as a '''Newtonian fluid with a constant velocity Vmax'''. Here, We neglect the heart-related jolts and turbulent flows caused by the cavities of the blood epithelium.<br><br />
<br />
The [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] moves along two axes. An X axis in the direction of blood flow and a Y axis orthogonal to the axis X.<br><br />
<br />
[[Image:RepèremouvementmécaniqueEn.png|center|400px]]<br><br />
<br />
=====The phage propagation in the bloodstream=====<br />
<br />
The movement '''in X''' depends solely on the '''propagation of phages''' the vessel due to blood flow. Indeed, we neglect the diffusion which takes place also along the X axis because it is 1000 times less than the propagation of particles in the blood (due to the importance of blood flow). The cellular carriers are moving at '''speeds spread on a dish''' from, Vmax in the center of the vessel at V0 against the vessel wall.<br><br />
<br />
[[Image:répartitionvitesseparaboliqueEN.png|center|500px]]<br><br />
<br />
The speed of phages decrease in approaching the vessel walls due to the friction forces which are opposing to the movement.<br><br />
<br />
We can determine how long the particle (with a Vmax velocity), ie the particles in the center of the ship, reached the end. This gives the time necessary to internalize all the phages of the bacteria.<br><br />
<br />
=====The diffusion through the walls of blood vessels=====<br />
<br />
The movement '''in Y''' is the distribution of phages in the blood (j(n)). It depends on the equation of diffusion of a particle (n) in a fluid (Fick's Law).<br><br />
<br />
[[Image:Equation diffusion du phage.png|center|170px]]<br><br />
<br />
With n the number of particles (phages), grad n the difference between the concentrations and D the diffusion coefficient.<br />
The cellular distribution of vectors within the blood vessel and then through the wall is a phenomenon of diffusion with output. So, there will always be a strong gradient of concentration of phage in the blood. We can therefore say that the gradient is constant (equal to 1) over time. Thus the diffusion rate (j(n)) is equal to D.<br />
<br />
<br />
=====Dispersal area of phage=====<br />
<br />
When we combine moving '''Y''' ('''diffusion rate''') and moving '''in X''' ('''blood flow velocity'''), we obtain, after integration on the '''perimeter of a blood vessel''', the action surface of [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]]. Then, we are able to determine the number of cancer cells per 100 [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] destroyed or 1 [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]].<br />
<br />
The diffusion rate of the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] is equal to 0.5 μm.s-1 and the size of a capillary blood is 10μm in diameter. The particle farthest places so 10s to reach the vessel wall.<br />
<br />
With this '''dissemination length''' (10s), the '''blood flow velocity''' (1x10 ^ 3μm.s-1) in the capillaries, and the surface of one cancer cell (1 micron square), we can determine:<br />
<br />
:* The '''length (L)''' covered by the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] released by one [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]].<br />
:* The '''surface (S)''' occupied by phages in blood vessel diameter of 2r.<br />
:* The amount of cancer cells available.<br />
<br />
L = 1 x 10^4 µm<br />
2r = 10 µm<br />
S = 2 x π x L r = 31.4 x 10^4 µm² <br />
<br />
Thus, a [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] can potentially target more than 31 000 cancer cells, yet it has that 100 [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]]. We can make a simplification to say that 100 [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] destroy 100 cancer cells. The efficiency of the dispersion is complete.<br />
<br />
For phage, once reached the wall, comes in the cellular internalization. This model responds to two courses of action.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
==== Step Five: The cell vector internalization ====<br />
<br />
Once in contact with the cell, the cell vector has two possible ways of action.<br />
<br />
:* The vector fix then it detaches from the cell.<br />
:* The vector fix then it is internalized within the cell. <br />
<br />
We can model this according time and the association constant (kon) and dissociation constant (koff) and internalization constant(Kint).<br />
<br />
This gives:<br />
[[Image : EqInt.jpg|center|280px]] <br><br />
<br />
With '''kon''' = 5.10^3 M-1s-1, '''koff''' = 8.10^-3 s-1 and '''Kint''' = 5,78.10^-4 s-1. If we calculate the '''global constant K'''', such as IDP = K' xt, we obtain '''K''''= 361.5 s-1. Thus more than 360 phages are internalized per second in contact with a wall.<br />
<br />
If we compare the time required to internalize a phage compared to waiting times before the cell enters apoptosis in response to the entrance of a vector cell (1 hour), it appears logical to neglect the internalization of cell vectors (IDP = constant = 360 phages/s) in the final equation. <br />
<br />
Thus, with a total efficiency of the phage diffusion, and a neglected internalization time, we can say that the efficiency constant '''λ is equal to 1'''. <br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
== Simultaneous evolution of DVS and tumor ==<br />
<br />
The evolution equation of our model over time is equal to:<br><br />
[[Image :EqFinaleEN.jpg|center|700px]][[Image:Bibou3.png|float|right|150px]]<br />
With :<br><br />
<div style="margin-left: 100px;"><br />
- Nc (t), the number of cancer cells depending time,<br><br />
- V (t), tumor volume,<br><br />
- V1 and V2, two tumor volumes respectively times t1 and t2, <br><br />
- Vcc, the volume of a cancer cell,<br><br />
- Nbi, the number of injected [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]],<br><br />
- Pp, the lung percentage of [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors]] relative to the injected dose,<br><br />
- DTB, the doubling time of [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]],<br><br />
- tinj, injection time of the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vectors],<br><br />
- Npl, the number of [[Team:SupBiotech-Paris/Concept2#drapeau|cell vectors]] released by [[Team:SupBiotech-Paris/Concept1Fr#drapeau|bacteria]].<br><br />
</div><br />
We can neglected (differences between the time or space scales) some factors: <br><br />
<div style="margin-left: 100px;"><br />
- Kdeg, the degradation constant of the phage, because all the phages were internalized in 10s.<br><br />
</div><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
<br />
== Treatment simulation ==<br />
<br />
The DVS need two injection:<br />
:* Injection of DVS,<br />
:* Injection of the activator, doxycycline.<br />
Here's a simulator, which calculates how long must we inject doxycycline to completely eliminate the tumor.<br><br />
<html><br />
<center><br />
<embed <br />
src="https://static.igem.org/mediawiki/2009/e/ec/SimulationSBP.swf" <br />
width="640"<br />
height="320"<br />
allowscriptaccess="always"<br />
allowfullscreen="true"<br />
/><br />
</center><br />
</html><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Treatement_modeling#drapeau|Back to top]]</span><br />
.<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Conclusion3#drapeau" target="_self"><br />
<img title="Lest's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Tissue_targetingTeam:SupBiotech-Paris/Tissue targeting2009-10-22T01:32:40Z<p>Aurel: /* Experimental method */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Tissue targeting =<br />
<br />
== Context ==<br />
<br />
The non-small cell lung cancer, or NSCLC, is a cancer, which develops in the organ lumen. This tumor localization is essentially due to factors triggering the tumorogenesis (example: tobacco). A logic route of administration for the treatment would be the aerosol route, though a nebulization of the bacteria. But, this route is not the one that we chose for our treatment. The DVS treatment applied to the lungs cancer is administrated by intravenous.<br><br />
<br />
== Objective ==<br />
<br />
We decided to check and quantify the <i>Mycobacterium avium</i> presence, the tissue vector, inside lungs, during the intraveinous administration. Thus, we realized an <i>in vivo</i> study in several murine models.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Experimental method ==<br />
<br />
To realize the <i>in vivo</i> study, we designed a bioluminescence protocol, which allows the real-time monitoring, of our tissue vector propagation in mice. The bioluminescence is a good reporter system to analyze the mycobacterial implantation and the clearance <i>in vivo</i>. We used a tool kindly provided by Dr Brian D. Robertson (Imperial College London researcher). This tool is a plasmid, once electroporated in M. <i>avium</i> can produce the firefly luciferase, a reporter gene, allowing the monitoring. Indeed, the luciferase catalysis the luciferin oxidation into oxiluciferin, involving photons emission, which can be caught by a CCD camera photosensitive (IVIS, Imaging System 50, Xenogen).<br><br />
Unfortunately, the growth of electroporated mycobacteria with the monitoring plasmid, was so slow, it was not possible to realize this study within the given time (generation time of M. <i>avium</i>: nearly 20 hours. That is why we decided to change our protocol.<br><br />
Following this lack of time, we used another protocol (detailed in the following subpart) to check and quantify the presence of our tissue vector in the lung. For this, we studied in the literature what dose of M.<i>avium</i> could be injected into a mouse model. Different studies, on several pathologies of bacterial origin, mentioned the injection of 105 to 107 CFU.ml-1 (colony forming units) per mouse by intravenous route.<br><br />
We chose to realize this experiment on 3 murine model: immunodepressed, normal, and cancerous.<br><br />
<br />
[[Image : 3modelmurinEn.png|center|650px]]<br />
<br />
According to several studies, it seems that mycobacteria has a specific affinity for fibronectins, surexpressed at the surface of tumor cells. Mycobacterial tropism for tumor area has not been yet demonstrated, the cancerous murine model allows us to test this hypothesis.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
=== Experimental protocol details ===<br />
<br />
:* Day -7: Inoculation of 4.10^6 LBS cells (fibroblastic cancer cells) subcutaneously on 2 black 6 mice.<br><br />
:* Day 0: Inoculation of 10^6 CFU.ml-1* of tissue vectors, M.avium, by IV route (intravenous) injected in the tail of the 3 mouse models: immunodepressed, cancer, and normal (2 mice per model).<br><br />
:: The concentration of injected tissue vectors was determined by measuring the optical density. The optical density of 10^6 bacteria is not detectable in a spectrophotometer. We therefore measured the cell suspension absorbance of 10^8 bacteria. Once diluted by 100, we obtained the desired concentration is: 10^6 bacteria.<br><br />
:* Day 7: Mice sacrifice, organs extraction and cell suspension obtention, from the lungs, liver, spleen.<br><br />
<br />
The cell suspension obtained is then analyzed by flow cytometry, to verify the size and granularity of cells. These 2 characteristics are totally different between eukaryotic cells comprising murine organs, and the vector tissue M.avium of prokaryotic origin.<br><br />
We can thus verify and quantify the presence of the vector tissue.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Results ==<br />
<br />
A lung sample of each murine models, has been analysed by flow cytometry, to determine and quantify our tissue vector presence, M.<i>avium</i> in the lungs. This experiment could not be repeated more than once, we can only be reserved for these results. Nevertheless, various publications have already demonstrated the <i>Mycobacterium avium</i> implantation in the lungs, thus proving the credibility and feasibility of this study.<br><br />
<br />
[[Image : Temoincyto.png|center|600px]]<br />
<br />
From the results above, we determined the signature of a sample of <i>Mycobacterium avium </i> by flow cytometry (red zone R1). The left image corresponds to background noise or signal of the cytometer. However, It would have been very interesting to make a 3rd control, with the acquisition of a sample of lungs of mice not infected with <i> M.avium </i>.<br><br />
<br />
[[Image : Echancyto1En.png|center|600px]]<br />
<br />
In the above samples, it is difficult to find specifically the signature of the « mycobacterium » sample. It is also very difficult to discern any difference between the 2 mouse model, Nod Scid and Black 6. <br />
<br />
[[Image : Echancyto12En.png|center|300px|float|left]]<br />
<br />
The opposite samples, does not allow us to determine a possible influence of the subcutaneous tumor on the mycobacterial tropism, or even to differentiate the <i>M. avium</i> tropism in this model, from others models.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Discussion ==<br />
<br />
This experiment achieved only a single time, failed to meet our goals, to identify and quantify the <i> M. avium </i> tropisms. However, lack of time is another factor to consider. Indeed, this analysis by flow cytometry was performed one week after the injection of <i> M. avium </i> in mice. After reading various publications, it is interesting to note that the significant data of <i>M. avium</i> implantation in the lungs, are obtained after many weeks. Therefore, it explains our difficulty to quantify by flow cytometry, the presence of the vector tissue after one week. <br><br />
<br />
[[Image : Graphpoumon.png|center|200px|float]]<br />
<br />
The above figure is extracted from the publication « [[Team:SupBiotech-Paris/Bibliography#revulung|Thymus as a target for mycobacterial infections]] ». A similar protocol was performed with injection of 10 ^ 6 CFU / ml <i> M. <i> avium </i> administered intravenously. . It is described in this figure that the lungs are infected from the first day of infection, but in very small amount (just over 10 ^ 1, then it is more prolonged with the rapid spread (10 ^ 1 to 10 ^ 6 CFU / organ in less than 5 weeks). This proves, therefore, that the lungs are colonized by M.<i> avium </i>.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Conclusion ==<br />
<br />
Although our experiment does not prove significantly the presence of <i>M. avium</i> in the lung, it has been proven many times that this type of bacteria colonizes the lung. This organ is a one of the natural tropisms of ''M. avium'' in mice and humans.<br><br />
We can use the [[Team:SupBiotech-Paris/Concept#DVS|DVS]] on lung cancer, because we are confident that the [[Team:SupBiotech-Paris/Concept1#drapeau|vector tissue]] can target the organism and release the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Antitumor_action#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Tissue_targetingTeam:SupBiotech-Paris/Tissue targeting2009-10-22T01:31:14Z<p>Aurel: /* Objective */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Tissue targeting =<br />
<br />
== Context ==<br />
<br />
The non-small cell lung cancer, or NSCLC, is a cancer, which develops in the organ lumen. This tumor localization is essentially due to factors triggering the tumorogenesis (example: tobacco). A logic route of administration for the treatment would be the aerosol route, though a nebulization of the bacteria. But, this route is not the one that we chose for our treatment. The DVS treatment applied to the lungs cancer is administrated by intravenous.<br><br />
<br />
== Objective ==<br />
<br />
We decided to check and quantify the <i>Mycobacterium avium</i> presence, the tissue vector, inside lungs, during the intraveinous administration. Thus, we realized an <i>in vivo</i> study in several murine models.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Experimental method ==<br />
<br />
To realize the <i>in vivo</i> study , we designed a bioluminescence protocol, which allows the real-time monitoring, of our tissue vector propagation in mice. The bioluminescence is a good reporter system to analyze the mycobacterial implantation and the clearance <i>in vivo</i>. We used a tool kindly provided by Dr Brian D. Robertson (Imperial College London researcher). This tool is a plasmid, once electroporated in M. <i>avium</i> can produce the firefly luciferase, a reporter gene, allowing the monitoring. Indeed, the luciferase catalysis the luciferin oxidation into oxiluciferin, involving photons emission, which can be caught by a CCD camera photosensitive (IVIS, Imaging System 50, Xenogen).<br><br />
Unfortunately, the growth of electroporated mycobacteria with the monitoring plasmid, was so slow, it was not possible to realize this study within the given time (generation time of M. <i>avium</i>: nearly 20 hours. That is why we decided to change our protocol.<br><br />
Following this lack of time, we used another protocol (detailed in the following subpart) to check and quantify the presence of our tissue vector in the lung. For this, we studied in the literature what dose of M.<i>avium</i> could be injected into a mouse model. Different studies, on several pathologies of bacterial origin, mentioned the injection of 105 to 107 CFU.ml-1 (colony forming units) per mouse by intravenous route.<br><br />
We chose to realize this experiment on 3 murine model: immunodepressed, normal, and cancerous.<br><br />
<br />
[[Image : 3modelmurinEn.png|center|650px]]<br />
<br />
According to several studies, it seems that mycobacteria has a specific affinity for fibronectins, surexpressed at the surface of tumor cells. Mycobacterial tropism for tumor area has not been yet demonstrated, the cancerous murine model allows us to test this hypothesis.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
=== Experimental protocol details ===<br />
<br />
:* Day -7: Inoculation of 4.10^6 LBS cells (fibroblastic cancer cells) subcutaneously on 2 black 6 mice.<br><br />
:* Day 0: Inoculation of 10^6 CFU.ml-1* of tissue vectors, M.avium, by IV route (intravenous) injected in the tail of the 3 mouse models: immunodepressed, cancer, and normal (2 mice per model).<br><br />
:: The concentration of injected tissue vectors was determined by measuring the optical density. The optical density of 10^6 bacteria is not detectable in a spectrophotometer. We therefore measured the cell suspension absorbance of 10^8 bacteria. Once diluted by 100, we obtained the desired concentration is: 10^6 bacteria.<br><br />
:* Day 7: Mice sacrifice, organs extraction and cell suspension obtention, from the lungs, liver, spleen.<br><br />
<br />
The cell suspension obtained is then analyzed by flow cytometry, to verify the size and granularity of cells. These 2 characteristics are totally different between eukaryotic cells comprising murine organs, and the vector tissue M.avium of prokaryotic origin.<br><br />
We can thus verify and quantify the presence of the vector tissue.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Results ==<br />
<br />
A lung sample of each murine models, has been analysed by flow cytometry, to determine and quantify our tissue vector presence, M.<i>avium</i> in the lungs. This experiment could not be repeated more than once, we can only be reserved for these results. Nevertheless, various publications have already demonstrated the <i>Mycobacterium avium</i> implantation in the lungs, thus proving the credibility and feasibility of this study.<br><br />
<br />
[[Image : Temoincyto.png|center|600px]]<br />
<br />
From the results above, we determined the signature of a sample of <i>Mycobacterium avium </i> by flow cytometry (red zone R1). The left image corresponds to background noise or signal of the cytometer. However, It would have been very interesting to make a 3rd control, with the acquisition of a sample of lungs of mice not infected with <i> M.avium </i>.<br><br />
<br />
[[Image : Echancyto1En.png|center|600px]]<br />
<br />
In the above samples, it is difficult to find specifically the signature of the « mycobacterium » sample. It is also very difficult to discern any difference between the 2 mouse model, Nod Scid and Black 6. <br />
<br />
[[Image : Echancyto12En.png|center|300px|float|left]]<br />
<br />
The opposite samples, does not allow us to determine a possible influence of the subcutaneous tumor on the mycobacterial tropism, or even to differentiate the <i>M. avium</i> tropism in this model, from others models.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Discussion ==<br />
<br />
This experiment achieved only a single time, failed to meet our goals, to identify and quantify the <i> M. avium </i> tropisms. However, lack of time is another factor to consider. Indeed, this analysis by flow cytometry was performed one week after the injection of <i> M. avium </i> in mice. After reading various publications, it is interesting to note that the significant data of <i>M. avium</i> implantation in the lungs, are obtained after many weeks. Therefore, it explains our difficulty to quantify by flow cytometry, the presence of the vector tissue after one week. <br><br />
<br />
[[Image : Graphpoumon.png|center|200px|float]]<br />
<br />
The above figure is extracted from the publication « [[Team:SupBiotech-Paris/Bibliography#revulung|Thymus as a target for mycobacterial infections]] ». A similar protocol was performed with injection of 10 ^ 6 CFU / ml <i> M. <i> avium </i> administered intravenously. . It is described in this figure that the lungs are infected from the first day of infection, but in very small amount (just over 10 ^ 1, then it is more prolonged with the rapid spread (10 ^ 1 to 10 ^ 6 CFU / organ in less than 5 weeks). This proves, therefore, that the lungs are colonized by M.<i> avium </i>.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
== Conclusion ==<br />
<br />
Although our experiment does not prove significantly the presence of <i>M. avium</i> in the lung, it has been proven many times that this type of bacteria colonizes the lung. This organ is a one of the natural tropisms of ''M. avium'' in mice and humans.<br><br />
We can use the [[Team:SupBiotech-Paris/Concept#DVS|DVS]] on lung cancer, because we are confident that the [[Team:SupBiotech-Paris/Concept1#drapeau|vector tissue]] can target the organism and release the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Tissue_targeting#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Antitumor_action#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-22T01:16:39Z<p>Aurel: /* Cancer cell line and reported gene */</p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the ability to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction which has been extracted from a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offers a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the adenovirus 5[[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its prefix and suffix. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair for genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure 1. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 11) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure 2 shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure 2). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragment coding for RGD sequence alone has a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Antitumoral Plasmide =<br />
<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing into tumor cells the wild type genes coding for functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] allowed us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés eng.jpeg|center]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
Cancer cell lines, which have a mutation of tumor suppressor gene has been selected from our database. We also possess a wild type version of the TP53 gene. Then we choose the prostatic cancer p53 mutated DU-145 in the goals to test if bringing the wild type version of the p53 protein (p53wt) in the DU-145 cell line allows to induce the apoptotic process. <br><br />
<br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The running of the study ==<br />
<br />
To analyse the timing of plasmid expression in the DU-145 cell line, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48 hours after the plasmid electroporation. By coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. We use a control without the plasmid pcDNA3 CMV+p53wt to quatify the level of death cells causes by the electroporation and by the culture transfert. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
[[image:planning eng.jpeg|center]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9 a.m, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 4 p.m, day 3). <br><br />
<br />
The first cytometric analyze allowed us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, allowed us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Results [1,2] ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let us to try a second experiment. <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53, cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin by the genome fragmentation observation, consequence to the CAD (Caspase Activated DNase) activity, a specific endonuclease of the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) by using similar analyses on a U251 cancer strain from a glioma. <br><br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the test population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA fragmentation :<br><br />
<br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a fragmentation of their genome, characteristic of the apoptosis process (laddering pattern).<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Concept#drapeau|DVS]], application in the fight against non small cell lung cancer, is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
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<a href="https://2009.igem.org/Team:SupBiotech-Paris/Treatement_modeling#drapeau" target="_self"><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-22T01:15:38Z<p>Aurel: /* Cancer cell line and reported gene */</p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the ability to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction which has been extracted from a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offers a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the adenovirus 5[[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its prefix and suffix. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair for genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure 1. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 11) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure 2 shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure 2). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragment coding for RGD sequence alone has a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Antitumoral Plasmide =<br />
<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing into tumor cells the wild type genes coding for functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] allowed us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés eng.jpeg|center]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
Cancer cell lines, which have a mutation of tumor suppressor gene has been selected from our databasis. We also possess a wild type version of the TP53 gene. Then we choose the prostatic cancer p53 mutated DU-145 in the goals to test if bringing the wild type version of the p53 protein (p53wt) in the DU-145 cell line allows to induce the apoptotic process. <br><br />
<br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The running of the study ==<br />
<br />
To analyse the timing of plasmid expression in the DU-145 cell line, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48 hours after the plasmid electroporation. By coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. We use a control without the plasmid pcDNA3 CMV+p53wt to quatify the level of death cells causes by the electroporation and by the culture transfert. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
[[image:planning eng.jpeg|center]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9 a.m, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 4 p.m, day 3). <br><br />
<br />
The first cytometric analyze allowed us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, allowed us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Results [1,2] ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let us to try a second experiment. <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53, cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin by the genome fragmentation observation, consequence to the CAD (Caspase Activated DNase) activity, a specific endonuclease of the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) by using similar analyses on a U251 cancer strain from a glioma. <br><br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the test population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA fragmentation :<br><br />
<br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a fragmentation of their genome, characteristic of the apoptosis process (laddering pattern).<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Concept#drapeau|DVS]], application in the fight against non small cell lung cancer, is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.<br><br />
<br />
<br />
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<br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-22T01:12:15Z<p>Aurel: /* Discussion */</p>
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<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the ability to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction which has been extracted from a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offers a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the adenovirus 5[[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its prefix and suffix. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair for genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure 1. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 11) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure 2 shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure 2). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragment coding for RGD sequence alone has a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Antitumoral Plasmide =<br />
<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing into tumor cells the wild type genes coding for functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] allowed us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés eng.jpeg|center]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
Cancer cell lines , which have a mutation of tumor suppressor gene has been selected from our databasis. We also possess a wild type version of the TP53 gene. Then we choose the prostatic cancer p53 mutated DU-145 in the goals to test if bringing the wild type version of the p53 protein (p53wt) in the DU-145 cell line allows to induce the apoptotic process. <br><br />
<br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The running of the study ==<br />
<br />
To analyse the timing of plasmid expression in the DU-145 cell line, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48 hours after the plasmid electroporation. By coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. We use a control without the plasmid pcDNA3 CMV+p53wt to quatify the level of death cells causes by the electroporation and by the culture transfert. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
[[image:planning eng.jpeg|center]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9 a.m, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 4 p.m, day 3). <br><br />
<br />
The first cytometric analyze allowed us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, allowed us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Results [1,2] ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let us to try a second experiment. <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53, cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin by the genome fragmentation observation, consequence to the CAD (Caspase Activated DNase) activity, a specific endonuclease of the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) by using similar analyses on a U251 cancer strain from a glioma. <br><br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the test population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA fragmentation :<br><br />
<br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a fragmentation of their genome, characteristic of the apoptosis process (laddering pattern).<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Concept#drapeau|DVS]], application in the fight against non small cell lung cancer, is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-22T01:02:50Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens celle du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]]. Ce dernier est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]], acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]]. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3' <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure 1. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] = 1715bp pour l’échantillon 11 et la de protéine D = 385bp pour l’échantillon 7 et 8. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (7 et 8) et de la base de penton (9, 10, 11) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure 2 montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (LambdaPb286-393), des phages lambda recombinés avec la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure 3). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes. Cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (fragment 1-571) car l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée des bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant la version saine des suppresseurs de tumeur non-fonctionnels.<br><br />
<br />
C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
<br />
[[image: gènes mutés.jpeg|center]]<br />
<br />
Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== L’objectif ==<br />
<br />
L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
<br />
== Démarche expérimentale ==<br />
<br />
<br />
=== Lignée cancéreuse et gène apporté ===<br />
<br />
Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Incorporation du gène TP53 ===<br />
<br />
L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
<br />
<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-22T00:57:17Z<p>Aurel: /* Experimental approach */</p>
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<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
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= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the ability to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction which has been extracted from a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offers a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the adenovirus 5[[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its prefix and suffix. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair for genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 11) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
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A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
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Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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= Antitumoral Plasmide =<br />
<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
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The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing into tumor cells the wild type genes coding for functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] allowed us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés eng.jpeg|center]]<br />
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These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
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== Experimental approach ==<br />
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=== Cancer cell line and reported gene ===<br />
<br />
Cancer cell lines , which have a mutation of tumor suppressor gene has been selected from our databasis. We also possess a wild type version of the TP53 gene. Then we choose the prostatic cancer p53 mutated DU-145 in the goals to test if bringing the wild type version of the p53 protein (p53wt) in the DU-145 cell line allows to induce the apoptotic process. <br><br />
<br />
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<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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=== TP53 gene incorporation ===<br />
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Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
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<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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=== Apoptosis detection ===<br />
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Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
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<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
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<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== The running of the study ==<br />
<br />
To analyse the timing of plasmid expression in the DU-145 cell line, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48 hours after the plasmid electroporation. By coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. We use a control without the plasmid pcDNA3 CMV+p53wt to quatify the level of death cells causes by the electroporation and by the culture transfert. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
[[image:planning eng.jpeg|center]] <br />
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Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9 a.m, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 4 p.m, day 3). <br><br />
<br />
The first cytometric analyze allowed us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, allowed us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Results [1,2] ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let us to try a second experiment. <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
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<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
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<br />
<br />
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<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53, cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin by the genome fragmentation observation, consequence to the CAD (Caspase Activated DNase) activity, a specific endonuclease of the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) by using similar analyses on a U251 cancer strain from a glioma. <br><br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the test population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA fragmentation :<br><br />
<br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
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<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a fragmentation of their genome, characteristic of the apoptosis process (laddering pattern).<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Concept#drapeau|DVS]], application in the fight against non small cell lung cancer, is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.<br><br />
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<a href="https://2009.igem.org/Team:SupBiotech-Paris/Treatement_modeling#drapeau" target="_self"><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-22T00:45:28Z<p>Aurel: /* The debate program */</p>
<hr />
<div>{{Template:Supbiotechcss14.css}}<br />
{{Template:SupbiotechparisEn2}}<br />
<br />
= Ethics =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debate program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
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<center><br />
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<a href="https://static.igem.org/mediawiki/2009/1/1d/Programme_of_ethic_debate1.pdf" target="_blank"><br />
<img title="Programme of Ethics Debate" style="width: 250px;" src="https://static.igem.org/mediawiki/2009/a/ae/Miniature_conf%C3%A9rence_ethique_en.png";><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions. François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working for the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of the power of fragility ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible compared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we are still very vulnerable and far to be perfect. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== Modification of the living representation ====<br />
<br />
Searching to synthesize and modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understanding and generating life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Problematic linked to applications ===<br />
<br />
==== Control of the evolution of synthetic biology products ====<br />
<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ».<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== The benefits/risks ratio ====<br />
<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== Intellectual properties ====<br />
<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Problematics related to the DVS project ===<br />
<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products. If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Conclusion ===<br />
<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
== Survey ==<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
*32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.<br><br />
<br />
<center>'''Do you think that patents slow innovation?'''</center><br />
[[Image:sondage breve = ralentissement innovation.png|center]]<br><br />
<br />
*As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. <br><br />
<br />
<center>'''Do you think an organism created by synthetic biology should be patented?'''</center><br />
[[Image:sondage brevetabilité d'un OGS.png|center|center]]<br><br />
<br />
<br />
Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? <br><br />
<br />
<br />
*50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. <br><br />
<br />
<center>'''As part of a therapeutic application, can we afford to create everything from the living?'''</center><br />
[[Image:sondage application thérapeutique.png|sondage application thérapeutique.png|center]]<br><br />
<br><br />
<br><br />
<br />
*<center>'''Do we risk diverging the evolution?'''</center><br />
[[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png|center]]<br />
<br />
Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. <br><br />
<br />
*Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.<br><br />
<center>'''Can we control the living?'''</center><br />
[[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png|center]]<br />
<br><br />
<br><br />
<br><br />
<br />
<br />
*<center>'''Do you think we should be able to control the spread of systems that we built'''</center><br />
[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png|center]]<br />
<br />
Faced with theses questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? <br><br />
<br />
*Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. <br><br />
<center>'''In the context of synthetic biology, is global governance feasible?'''</center><br />
[[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png|center]]<br />
<br />
<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-22T00:39:20Z<p>Aurel: /* Summarization of reflections */</p>
<hr />
<div>{{Template:Supbiotechcss14.css}}<br />
{{Template:SupbiotechparisEn2}}<br />
<br />
= Ethics =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
<html><br />
<center><br />
<div style=""><br />
<a href="https://static.igem.org/mediawiki/2009/1/1d/Programme_of_ethic_debate1.pdf" target="_blank"><br />
<img title="Programme of Ethics Debate" style="width: 250px;" src="https://static.igem.org/mediawiki/2009/a/ae/Miniature_conf%C3%A9rence_ethique_en.png";><br />
</a></div><br />
</center><br />
</html><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
== Discover the videos of the debate ! ==<br />
<br />
<html><br />
<right><br />
<p align="center"><br />
<object width="600"><param name="movie" value="http://www.youtube.com/v/wPVe1pruUQA&hl=fr&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/wPVe1pruUQA&hl=fr&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
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<html><br />
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<p align="center"><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/-OdjR2Z9Sfs&hl=fr&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/-OdjR2Z9Sfs&hl=fr&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</p><br />
</html><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions. François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working for the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of the power of fragility ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible compared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we are still very vulnerable and far to be perfect. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== Modification of the living representation ====<br />
<br />
Searching to synthesize and modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understanding and generating life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Problematic linked to applications ===<br />
<br />
==== Control of the evolution of synthetic biology products ====<br />
<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ».<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== The benefits/risks ratio ====<br />
<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
==== Intellectual properties ====<br />
<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Problematics related to the DVS project ===<br />
<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products. If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
=== Conclusion ===<br />
<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
== Survey ==<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
*32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.<br><br />
<br />
<center>'''Do you think that patents slow innovation?'''</center><br />
[[Image:sondage breve = ralentissement innovation.png|center]]<br><br />
<br />
*As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. <br><br />
<br />
<center>'''Do you think an organism created by synthetic biology should be patented?'''</center><br />
[[Image:sondage brevetabilité d'un OGS.png|center|center]]<br><br />
<br />
<br />
Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? <br><br />
<br />
<br />
*50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. <br><br />
<br />
<center>'''As part of a therapeutic application, can we afford to create everything from the living?'''</center><br />
[[Image:sondage application thérapeutique.png|sondage application thérapeutique.png|center]]<br />
<br />
*<center>Do we risk diverging the evolution?</center><br />
<br />
Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. <br><br />
<br />
[[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png|center]]<br />
<br />
<center>'''Can we control the living?'''</center><br />
[[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png|center]]<br />
<br />
*Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.<br><br />
<center>'''Do you think we should be able to control the spread of systems that we built'''</center><br />
[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png|center]]<br />
<br />
*Faced with theses questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? <br><br />
<br />
*Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. <br><br />
<center>'''In the context of synthetic biology, is global governance feasible?'''</center><br />
[[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png|center]]<br />
<br />
<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ethic#drapeau|Back to top]]</span><br />
<br />
<br />
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<a href="https://2009.igem.org/Team:SupBiotech-Paris/Safety#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-22T00:01:55Z<p>Aurel: /* Problematics related to the DVS project */</p>
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<br />
<br />
= Ethic =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
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<center><br />
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<a href="https://static.igem.org/mediawiki/2009/1/1d/Programme_of_ethic_debate1.pdf" target="_blank"><br />
<img title="Programme of Ethics Debate" style="width: 250px;" src="https://static.igem.org/mediawiki/2009/a/ae/Miniature_conf%C3%A9rence_ethique_en.png";><br />
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== Discover videos of the debate ! ==<br />
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<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement itself of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions . François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working to the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of fragility power ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible copared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we still very vulnerable and far to be perfect. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Modification of the living representation ====<br />
<br />
Search to synthesize and to modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understand and generate life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problematic linked to applications ===<br />
<br />
==== Evolution control synthetic biology products ====<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ». <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== The benefits/risks ratio ====<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Intellectual properties ====<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problematics related to the DVS project ===<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products / / / / If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br />
<br><span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Conclusion ===<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
<br />
== Survey ==<br />
<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
*32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.[[Image:sondage breve = ralentissement innovation.png]]<br />
*As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. [[Image:sondage brevetabilité d'un OGS.png]]<br />
*Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? [[Image:sondage application thérapeutique.png|sondage application thérapeutique.png]]<br />
*50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. [[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png]]<br />
*Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. [[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png]]<br />
*Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png]]<br />
*Faced with these questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? <br />
*Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. [[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png]]<br />
<br />
<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Safety#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-22T00:01:22Z<p>Aurel: /* Intellectual properties */</p>
<hr />
<div>{{Template:Supbiotechcss14.css}}<br />
{{Template:SupbiotechparisEn2}}<br />
<br />
<br />
<br />
= Ethic =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
<html><br />
<center><br />
<div style=""><br />
<a href="https://static.igem.org/mediawiki/2009/1/1d/Programme_of_ethic_debate1.pdf" target="_blank"><br />
<img title="Programme of Ethics Debate" style="width: 250px;" src="https://static.igem.org/mediawiki/2009/a/ae/Miniature_conf%C3%A9rence_ethique_en.png";><br />
</a></div><br />
</center><br />
</html><br />
<br />
== Discover videos of the debate ! ==<br />
<br />
VIDEOS<br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement itself of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions . François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working to the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of fragility power ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible copared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we still very vulnerable and far to be perfect. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Modification of the living representation ====<br />
<br />
Search to synthesize and to modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understand and generate life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problematic linked to applications ===<br />
<br />
==== Evolution control synthetic biology products ====<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ». <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== The benefits/risks ratio ====<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Intellectual properties ====<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problematics related to the DVS project ===<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products / / / / If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
=== Conclusion ===<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
<br />
== Survey ==<br />
<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
*32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.[[Image:sondage breve = ralentissement innovation.png]]<br />
*As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. [[Image:sondage brevetabilité d'un OGS.png]]<br />
*Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? [[Image:sondage application thérapeutique.png|sondage application thérapeutique.png]]<br />
*50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. [[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png]]<br />
*Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. [[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png]]<br />
*Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png]]<br />
*Faced with these questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? <br />
*Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. [[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png]]<br />
<br />
<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Safety#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-22T00:01:00Z<p>Aurel: /* The benefits/risks ratio */</p>
<hr />
<div>{{Template:Supbiotechcss14.css}}<br />
{{Template:SupbiotechparisEn2}}<br />
<br />
<br />
<br />
= Ethic =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
<html><br />
<center><br />
<div style=""><br />
<a href="https://static.igem.org/mediawiki/2009/1/1d/Programme_of_ethic_debate1.pdf" target="_blank"><br />
<img title="Programme of Ethics Debate" style="width: 250px;" src="https://static.igem.org/mediawiki/2009/a/ae/Miniature_conf%C3%A9rence_ethique_en.png";><br />
</a></div><br />
</center><br />
</html><br />
<br />
== Discover videos of the debate ! ==<br />
<br />
VIDEOS<br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement itself of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions . François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working to the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of fragility power ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible copared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we still very vulnerable and far to be perfect. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Modification of the living representation ====<br />
<br />
Search to synthesize and to modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understand and generate life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problematic linked to applications ===<br />
<br />
==== Evolution control synthetic biology products ====<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ». <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== The benefits/risks ratio ====<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
==== Intellectual properties ====<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span> <br />
=== Problematics related to the DVS project ===<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products / / / / If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
=== Conclusion ===<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
<br />
== Survey ==<br />
<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
*32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.[[Image:sondage breve = ralentissement innovation.png]]<br />
*As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. [[Image:sondage brevetabilité d'un OGS.png]]<br />
*Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? [[Image:sondage application thérapeutique.png|sondage application thérapeutique.png]]<br />
*50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. [[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png]]<br />
*Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. [[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png]]<br />
*Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png]]<br />
*Faced with these questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? <br />
*Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. [[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png]]<br />
<br />
<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Safety#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-21T23:58:07Z<p>Aurel: /* Evolution control synthetic biology products */</p>
<hr />
<div>{{Template:Supbiotechcss14.css}}<br />
{{Template:SupbiotechparisEn2}}<br />
<br />
<br />
<br />
= Ethic =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
<html><br />
<center><br />
<div style=""><br />
<a href="https://static.igem.org/mediawiki/2009/1/1d/Programme_of_ethic_debate1.pdf" target="_blank"><br />
<img title="Programme of Ethics Debate" style="width: 250px;" src="https://static.igem.org/mediawiki/2009/a/ae/Miniature_conf%C3%A9rence_ethique_en.png";><br />
</a></div><br />
</center><br />
</html><br />
<br />
== Discover videos of the debate ! ==<br />
<br />
VIDEOS<br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement itself of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions . François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working to the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of fragility power ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible copared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we still very vulnerable and far to be perfect. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Modification of the living representation ====<br />
<br />
Search to synthesize and to modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understand and generate life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problematic linked to applications ===<br />
<br />
==== Evolution control synthetic biology products ====<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ». <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== The benefits/risks ratio ====<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
==== Intellectual properties ====<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span> <br />
=== Problematics related to the DVS project ===<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products / / / / If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
=== Conclusion ===<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
<br />
== Survey ==<br />
<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
#32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.[[Image:sondage breve = ralentissement innovation.png]]<br />
#As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. [[Image:sondage brevetabilité d'un OGS.png]]<br />
#Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? [[Image:sondage application thérapeutique.png|sondage application thérapeutique.png]]<br />
#50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. [[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png]]<br />
#Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. [[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png]]<br />
#Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png]]<br />
#Faced with these questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? [[Image:.png|.png]]<br />
#Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. [[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png]]<br />
<br />
<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Safety#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-21T23:55:56Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss14.css}}<br />
{{Template:SupbiotechparisEn2}}<br />
<br />
<br />
<br />
= Ethic =<br />
<br />
The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
<html><br />
<center><br />
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== Discover videos of the debate ! ==<br />
<br />
VIDEOS<br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement itself of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions . François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working to the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of fragility power ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible copared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we still very vulnerable and far to be perfect. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Modification of the living representation ====<br />
<br />
Search to synthesize and to modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understand and generate life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
==== Evolution control synthetic biology products ====<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum observe that the pression responsible of the evolution will exist even for organisms which are not due to this pression; and that « it is more performant and less nocive that will go out of this; because these presion will stay ». <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Bioterrorists drifts ====<br />
<br />
The loss of control of living systems syntheticaly created could be intentional. The synthetic biology and the diffusion of knowledge that it put at disposal of a large public of genomes, notably pathogenes can be modified at low cost. In the case of our DVS project,some changes could transform our vector in biologic weapon like mentionned François le Fèvre: « we can imaginethat instead of target a cancer, we target neurons to send drogues that permit to weaken someone ». From 2003, a CIA report mentionned risks linked to live science development and the difficulty to limit the bioterrorism developement. It is necessary to limit access to data at the risk of slowing down progress of the knowledge in synthetic biology? <br><br />
<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== The benefits/risks ratio ====<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
==== Intellectual properties ====<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span> <br />
=== Problematics related to the DVS project ===<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products / / / / If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
=== Conclusion ===<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
<br />
== Survey ==<br />
<br />
<br />
Today everything is patented or patentable, and worse it is possible to patent in simple concepts that have not been applied. Thus the purchase, exchange, submission and management of the patents bank of a company is a real business activity and it can be really profitable. Patent an invention, a concept or a brand is there real consequences on the daily progress? That is what we asked students to respond Sup'Biotech.<br><br />
<br />
<br />
#32% believe that patents represent a barrier to innovation, while 43% disagreed. The opinion seems pretty divided, which is quite surprising because in theory the patent is a tool for encouraging innovation. Indeed, the temporary monopoly allows to finance investment in R & D. However, in practice the patent appears as a secondary tool, some do not even have little confidence, while others do not hesitate to follow the example of the law fragmentation when innovations are cumulative and / or complementary as computing, biotechnology or electronics.[[Image:sondage breve = ralentissement innovation.png]]<br />
#As part of a therapeutic application, we may wonder if we can patent a living thing, giving it a value? This is the question that is facing synthetic biology. [[Image:sondage brevetabilité d'un OGS.png]]<br />
#Like other technologies, synthetic biology would show us a new era, that of "Biolithic", where the living is becoming the tool. A tool that could be greatly promising to cure many diseases. But what is the therapeutic goal legislates she use? Synthetic biology thus challenges our life conception. Where is the boundary between natural and artificial? Can we afford to create everything from the living? Evolution can be "diverged"? [[Image:sondage application thérapeutique.png|sondage application thérapeutique.png]]<br />
#50% of students tend to reject this possibility of free manipulation with therapy pretext, however, 31% would consider it and 19% of students are wondering. As for a drift of evolution, 50% of students are quite convinced that evolution cannot be compromised by synthetic biology, however, 31% of students disagreed. [[Image:sondage divergence de l'évolution.png|sondage divergence de l'évolution.png]]<br />
#Researchers must ask themselves these questions and beware of unethical uses that could be made of such technologies, even for the purpose of curing diseases; this fear of a student speaks to the questions raised by the living instrumentalization facing synthetic biology. [[Image:sondage peut-on contrôler le vivant.png|sondage peut-on contrôler le vivant.png]]<br />
#Indeed, each advanced biological research contains a lot of questions on the health implications, environmental, social and ethical implications of possible applications of these discoveries. Are we able to control the living? Are we able to control the spread of systems that we built? While they are a majority think that researchers are capable of manipulating life, we remain skeptical with control its spread.[[Image:sondage maitriser la propagation des systèmes construits.png|sondage maitriser la propagation des systèmes construits.png]]<br />
#Faced with these questions, how the company will position itself and how to respect the ethics rules. Because of the life control, the public is faced with a control by research area while having the feeling of being dispossessed of research results. How the company is going to express their wishes on these issues? [[Image:.png|.png]]<br />
#Given the stakes, the debate should be pluralist and collective, we have to know who will control and how? Do we need new regulations, while those for existing GMOs are already far from perfection and unaccepted? Can we aspire to global governance? 46% of students believe that such governance is possible, while 31% think otherwise. [[Image:sondage gouvernance mondiale.png|sondage gouvernance mondiale.png]]<br />
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<br />
The survey draws the attention of politics, researchers and lawyers, reminding them that the innovation and therapeutic goal arguments are often wrongly used by supporters of a world where everything is protected and deposits. A collective discussion should take place to decide together how to maximize the positive applications of these technologies while minimizing the abusive risks.<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/EthicTeam:SupBiotech-Paris/Ethic2009-10-21T23:40:39Z<p>Aurel: </p>
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= Ethic =<br />
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The international competition iGEM gathering each year together more and more teams (110 teams for the 2009 session) added to 18 Europeans programs, 70 industries, 10000 laboratories in the world which have all the same common objective: the construction of living systems, following the assembly principle of functional modules. <br><br />
<br />
<br />
The emergence and the fast development of this discipline require reflection, to put a regulation system in place ready in the next 5 to 10 years for safe practices. <br><br />
Thus at the occasion of the iGEM concourse, we realized this debate to think about ethic stake linked to synthetic biology. <br><br />
<br />
== The debate program ==<br />
<br />
Debat program : <br><br />
<br />
#Introduction to synthetic biology, François Le Fèvre<br><br />
#Introduction to the Double Vectorization System (DVS) project developed by the team<br><br />
#Round table leaded by Thierry Magnin, and the Sup’Biotech Paris team: <br><br />
#* Synthetic biology / DVS Project - Formulation of risks and benefits: what are the risks, can we get round them, what are the effects on Human, animal and environment, the advantages of this discipline, where stop science and where start creation? The populations fears... <br><br />
#*Regulation, Access and right : at which point the knowledge should be protected, put in advance the « non patent » concept as well as regulations... <br><br />
<br />
<br />
Or linked program = Click to enlarge + video <br><br />
<br />
== Discover videos of the debate ! ==<br />
<br />
VIDEOS<br />
<br />
== Summarization of reflections ==<br />
<br />
« Ethic is the movement itself of the Liberty which searches a well life, in the solicitude toward others is in just use of social institutions »; Paul Ricoeur quotation, philosopher of the 20th century. In other terms, ethic represents the philosophical field gathering moral values which define the way we have to behave. <br><br />
<br><br />
Applied to synthetic biology, ethic indicates the way to follow to allow this discipline development by avoiding its drifts. Indeed, even if it lets dream to large perspectives like clean energy sources, accessible therapies to all or biological remediation methods, to manipulate the living rises regularly to a certain number of ethic questions . François Le Fèvre mentions « it is the first time that human is confronted to the possibility to create new forms of life ». <br><br />
<br><br />
It seemed important to us to interest to these points, beside the biologic engineering technic aspect. In this way, we organized an ethic debate based on the topic of the synthetic biology, in which some different expert key figures of the domain were invited. During this debate, different problematics were raised. Like emphasized Thierry Magnin, some of them are of metaphysical order, and concern notably what «this gives us as the living representation, as life »; some others concern direct applications and their technical aspects which can push us to imitate them. At the occasion of this debate, we presented our project to our guests in order to take out ethic questions.<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
=== Metaphysic problematics ===<br />
<br />
==== Aim of the synthetic biology ====<br />
It convinces first to interest in finality of this science. What are we trying to do? Are we looking to reach a perfection state? When we are working to the improvement of a living organism, in addition to technical difficulties, we have to ask if what are we doing is desirable. Without the egocentric drifts we can easily imagine, we could try to correct our weaknesses, handicap, diseases. Dorothée Benoit Browaeys put in advance that the context can change a « tare » in asset: « there are diseases which give you certain advantages. So to take up the titer of Alain Gras’ book on the fragility of the power, we could speak of fragility power ». <br><br />
<br><br />
However, potentials advantages seem sometimes negligible copared to the handicap: it is for example the case when we are affected by the HIV. And the engendered disease will not be controled, in Willy Rozenbaum opinion, « if we are not using synthetic biology ». More generally, this last one does not imagine « how we could do without it if we want to go towards an improvement of the human condition». The perfection myth seems not to worry him, because he affirms that we still very vulnerable and far to be perfect. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
==== Modification of the living representation ====<br />
<br />
Search to synthesize and to modify fundamentally organisms push to wonder about the definition itself of the living. Craig Venter affirms that « we pass from the capacity to read our genetic code to the capacity to write it». But understand and generate life mechanism can demistify it; and the fact to create living machines, in a precise goal, risk to give us a determinist vision of the living. Thierry Magnin wonders « in a context where life is assemble with bricks, what is doing the real difference between vegetal machinery, animal machinery and human machinery? ». After all, we can consider the difference between the three does not come from interactions between « bricks » which compose them. « How can I recognize a certain dignity of Living if all is built by blocs » ? <br><br />
<br><br />
Synthetic biology can reveal a play aspect, and this aspect can alter the respect that we carry to living organism : to quote one more time Thierry Magnin, « Those with what I am used to play, I often have difficulties to respect it». We can create « pieces » of living organism without of their context, stock, reproduce, transmit and assemble them. If we create biologic systems like we assemble “legos”, do not we risk considering living organisms, whose human, like simple assembling of pieces? And in this case, the respect that we consider to have face to them can be altered. Of course, we can consider that our creations are only biologic engines, synthetic distinctive machines of « natural » life forms. <br><br />
<br><br />
But where is the limit between these ones and the artificial life? The way of one and the other were created change their natures? It is however necessary to qualify the impact what biological synthesis could have on the way we consider life: how reminded François Le Fèvre, when «we synthesized urea, the first organic synthetic molecule, it has an entire debate to know if we created life or not»; and, how emphases Lluis Mir, we could ask same questions at the beginning of of chemistry. Two hundred years later, it can make smile. <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
=== Problamatic linked to applications ===<br />
<br />
==== Evolution control synthetic biology products ====<br />
Synthetic biology leads to the creation of living organisms which should not have exist without the human intervention and are not the fruit of a natural evolution. Will be able to control it? We are not controlling mechanism of the information storage in the living world, and we are far to be able to predict how will behave a group from its separate elements. We create parts, but will be able to predict emergent properties of their assembling? Furthermore, synthetics organisms, because they are living, evolve; will we be, asked Thierry Magnin, « in measure to control propagation of these lively engines that we construct? » Thanks to their capacity to evolve, do they risk to escape to our control? Willy Rozenbaum a fait remarquer que la pression responsable de l’évolution existera même pour les organismes qui ne lui sont pas dus a cette pression; et que « c’est le plus performant et le moins nocif qui va finalement sortir de cela ; parce que cette pression-là va rester ». <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
==== Dérives bioterroristes ====<br />
<br />
La perte de contrôle de systèmes vivants crées syntheticment pourrait être intentionnelle. La biology synthetic et la diffusion des connaissances qu’elle apporte met à disposition d’un large public des génomes, notamment des pathogènes pouvant être modifié à un coût relativement bas. Dans le cas de notre projet DVS, quelques changements pourraient transformer notre vecteur en arme biologique comme l’a mentionné François le Fèvre: « on peut imaginer qu’au lieu de cibler un cancer, on cible des neurones pour envoyer des drogues qui permettent d’affaiblir la personne ». Dès 2003, un rapport de la CIA a évoqué les risques liés au développement des sciences du vivant et la difficulté de limiter le développement du bioterrorisme. Faut-il limiter l’accès aux données sous peine de ralentir la progression des connaissances en biology synthetic ? <br><br />
<br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
==== The benefits/risks ratio ====<br />
To assess the risks and benefits of a science, we have to wonder for what it is intended, and if the risks are taken by beneficiaries. In the case of synthetic biology, risks are taken by the society and it must be the same for benefits. The financial interest of a small community does not have to harm the majority. Currently, the scientific community manages synthetic biology, but some applications, provided to generate significant revenues, might be developed despite the nuisance they cause. Therefore, as stated by Lluis Mir, "it remains the vision of science and society, and not markets." It is also important that involved researchers retain their critical thinking and continue to communicate the progress of their knowledge even if they work in an industrial or commercial context. <br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
==== Intellectual properties ====<br />
Thinking about the intellectual property of our project. We wanted that our treatment could be available at the lowest price. In this context, we asked about the open source development or patenting at least a part? The first option would allow any company to develop and improve it, but a private company could then patent a more rounded version of it, and impose prices that benefit the most. Furthermore, Willy Rozenbaum confirmed us that the clinical development would be very difficult to finance, "if you can convince a manufacturer to begin the preclinical tests, you will already have protected your model because otherwise you will not find manufacturers to develop it. " This last point would be less problematic with the second option as the funds generated by a patent would help persuading manufacturers, but access to data would be much more limited.<br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span> <br />
=== Problematics related to the DVS project ===<br />
One of the objective of this meeting was to discuss some issues related to our project DVS. The general points have been mentioned above, since these point apply to the whole synthetic biology. Specifically, we examined relative risks underlying the introduction of potentially pathogenic agents in the organism. <br><br />
<br><br />
Let’s begin with the importance of this risk. Mycobacterium avium is sometimes responsible for serious infections in humans. But, as noted by Willy Rozenbaum, "it is a bacterium that is ubiquitous, it is found in tap water, we are almost all contaminated" but this contamination has rarely consequential effects. The cases reported involved immunodepressed patients, for example. We also planned to analyze the effects of infection on tumors. Anyway, Willy Rozenbaum believes that "all that is not very annoying”. In addition to numerous tests and simulations that have to be conducted before the use of our treatment, this statement is justified by the fact that bacteria are lysed when there is a release of the phage, it does not persist in the body. <br><br />
Francois Le Fevre has legitimately questioned about the possibility that the phage infect other bacteria already present in the organism. We have therefore explained to him that our cell vector encapsidate only the therapeutic plasmid, not its genome. If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid, and the phage will not be able to multiply We can also worry about the drifts, and abuses of the transgene integration, as the risk of homologous recombination or risky integration. Lluis M. Mir supported us about this idea, that our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world": "there is no possible integration. That's the real advantage of being at the crossroads between eukaryotic and prokaryotic. <br><br />
<br><br />
Furthermore, Willy Rozenbaum reminded "this type of subject is very well controlled today in terms of security": the product would obviously not be marketed until being subjected to numerous tests to check its innocuousness. Organizations as Afssaps, in France manage the safety of health products / / / / If we consider that the risk is not negligible, we must ask whether it is worthwhile to be taken into account. Thierry Magnin gave a translation of the principle of responsibility made by Hans Jonas: "Before trying to estimate the risk, I'll try to work up on the most serious risk." Does the targeted disease justify it? According to Bernard Baertschi, "Cancer is an extremely serious disease, for which we accept to take risks even now." Francois Le Fevre acknowledged: "Anyway, if I have lungs cancer, I think I should take your medicine...” To conclude this section, we can quote Bernard Baertschi again: "We can take a risk if the person consents and if there is an expected benefit. <br><br />
<br><br />
<span style="float: right">[[Team:SupBiotech-Paris/Introduction1Fr#drapeau|Haut de page]]</span><br />
<br />
=== Conclusion ===<br />
Synthetic biology can become a very powerful tool if it remains under control. Risks exist, of course, but some causes for which it is an asset that justifies the taking. It is without doubt the scientific community to make the community accept this idea, by transmitting the knowledge. Some problems, such as various diseases, seem also to be resolved through it. But the sought interests are those of the entire society, and not particular groups. It might be beneficial to put quickly in place a regulation to avoid abuses, without limiting the development of this promising science<br><br />
<br />
<br />
== Survey ==<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Synthetic Biology#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Safety#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Conclusion3Team:SupBiotech-Paris/Conclusion32009-10-21T20:16:37Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
<br />
= Conclusion =<br />
<br />
<br />
Non-small cell lung cancer, or NSCLC, is an aggressive cancer. It grows in the lumen of the organ at high speed. This localization is highly correlated to the localization of factors launching the tumorgenesis (contained in the tobacco smoke for example). Tumor cells lose their apoptotic capacity after the functional loss of different tumor suppressors playing a role in the apoptotic pathway. The [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] application in the fight against cancer is based on the fact to reactivate these apoptotic pathway by bringing into tumor cells a wild-type version of genes coding for an healthy version of a non-functional tumor suppressor gene. The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] of [http://www.sanger.ac.uk/ Sanger institute] allow us to inventory genes the most likely to be mutated in the case of lung cancer. So, we had to determine the [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic plasmid]] composition applied to these physiopathology. <br><br />
<br />
<br />
Several studies showed that lung is one of the <i>Mycobactérium avium</i> natural tropism in mouse and Human. After an intravenous injection of <i>M. avium</i>, the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] gets to the lung and can grow inside it. So, the first [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] concept, for these application, is validated. For the second one, literature shows that the creation of a recombined phage with an adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]], a protein which allows to pass through membranes by endocytosis, is possible. The [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] creation, which has for objective to integer the [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic plasmid]] into eukaryotic tumor cells, is feasible. It is proved that we can modulate the efficiency of the interaction of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with integrins of eukaryotic cells by using the whole protein or only its RGD sequence alone. In the [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] project context it is the entire structure of the protein that we placed on the Lambda phage capsid. Because c’est l’ensemble de sa structure que l’on a placé sur la capside du phage lambda. Possessing a lower affinity for cell than if we used the RGD motif alone, [[Team:SupBiotech-Paris/Concept2#drapeau| cell vectors]] could more scatter and consequently, touch a higher number of cells.<br><br />
<br />
<br />
<i>In vivo</i> studies on human proved that to bring a wild-type version of a tumor suppressor gene into tumor cell for which the gene is mutated allow the launching of the apoptosis process and the inhibition of the tumor growth. <br><br />
<br />
<br />
By taking up all these data, it appears obvious that the [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] application in the anticancer fight against non small cell lung cancer represent a straight alternative to existing treatments, the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] targeting the organ of interest and the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] could deliver the [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic plasmid]] which apoptotic activity is confirmed. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Conclusion3#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Biobricks#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Conclusion3Team:SupBiotech-Paris/Conclusion32009-10-21T20:15:50Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
Non-small cell lung cancer, or NSCLC, is an aggressive cancer. It grows in the lumen of the organ at high speed. This localization is highly correlated to the localization of factors launching the tumorgenesis (contained in the tobacco smoke for example). Tumor cells lose their apoptotic capacity after the functional loss of different tumor suppressors playing a role in the apoptotic pathway. The [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] application in the fight against cancer is based on the fact to reactivate these apoptotic pathway by bringing into tumor cells a wild-type version of genes coding for an healthy version of a non-functional tumor suppressor gene. The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] of [http://www.sanger.ac.uk/ Sanger institute] allow us to inventory genes the most likely to be mutated in the case of lung cancer. So, we had to determine the [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic plasmid]] composition applied to these physiopathology. <br><br />
<br />
<br />
Several studies showed that lung is one of the <i>Mycobactérium avium</i> natural tropism in mouse and Human. After an intravenous injection of <i>M. avium</i>, the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] gets to the lung and can grow inside it. So, the first [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] concept, for these application, is validated. For the second one, literature shows that the creation of a recombined phage with an adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]], a protein which allows to pass through membranes by endocytosis, is possible. The [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] creation, which has for objective to integer the [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic plasmid]] into eukaryotic tumor cells, is feasible. It is proved that we can modulate the efficiency of the interaction of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with integrins of eukaryotic cells by using the whole protein or only its RGD sequence alone. In the [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] project context it is the entire structure of the protein that we placed on the Lambda phage capsid. Because c’est l’ensemble de sa structure que l’on a placé sur la capside du phage lambda. Possessing a lower affinity for cell than if we used the RGD motif alone, [[Team:SupBiotech-Paris/Concept2#drapeau| cell vectors]] could more scatter and consequently, touch a higher number of cells.<br><br />
<br />
<br />
<i>In vivo</i> studies on human proved that to bring a wild-type version of a tumor suppressor gene into tumor cell for which the gene is mutated allow the launching of the apoptosis process and the inhibition of the tumor growth. <br><br />
<br />
<br />
By taking up all these data, it appears obvious that the [[Team:SupBiotech-Paris/Introduction1#DVS|DVS]] application in the anticancer fight against non small cell lung cancer represent a straight alternative to existing treatments, the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] targeting the organ of interest and the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] could deliver the [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic plasmid]] which apoptotic activity is confirmed. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Conclusion3#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Biobricks#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Conclusion3FrTeam:SupBiotech-Paris/Conclusion3Fr2009-10-21T20:01:25Z<p>Aurel: /* Conclusion */</p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Conclusion =<br />
<br />
<br />
Le cancer du poumon non à petites cellules, ou NSCLC, est un cancer dit agressif. Il se développe dans la lumière de l’organe à une vitesse relativement élevée. Cette localisation tumorale est fortement corrélée à la localisation des facteurs déclenchant la tumorogénèse (contenus dans la fumée du tabac par exemple). Les cellules tumorales perdent leur capacité apoptotique suite à la perte fonctionnelle de divers suppresseurs de tumeur jouant un rôle dans la voie de signalisation de la cascade apoptotique. L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant une version saine des suppresseurs de tumeur non-fonctionnels. Le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] nous a permis de recenser les gènes les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon. Ainsi nous avons pu déterminer la composition du [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] appliqué à cette physiopathologie. <br><br />
<br />
<br />
De nombreuses études ont montrées que le poumon est l'un des tropismes naturels de <i>Mycobactérium avium</i> chez la souris, comme chez l’homme. Après une injection en intraveineuse de <i>M. avium</i>, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] arrivera bien au poumon et pourra s’y développer. Le premier concept du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], pour cette mise en application, est donc validé. Concernant le second, la littérature montre que la création d’un phage recombiné avec [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus, une protéine permettant le passage des membranes par endocytose, est possible. La création du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], qui aura pour objectif d’intégrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] au sein de cellules malades eucaryotes, est donc réalisable. Il est prouvé que l’on peut jouer sur l’efficacité d’interaction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] avec les intégrines des cellules eucaryotes en utilisant soit l’ensemble de sa structure soit son motif RGD seul. Dans le contexte du projet [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], c’est l’ensemble de sa structure que l’on a placé sur la capside du phage Lambda. Possédant alors une moins forte affinité pour les intégrines cellulaires que si on avait utilisé le motif RGD seul, les [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteurs cellulaires]] pourront d’avantage se disperser et par conséquence, toucher un plus grand nombre de cellules.<br><br />
<br />
<br />
Des études <i>in vivo</i> chez l’homme ont prouvées qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui ce gène est muté permet le déclenchement du processus d’apoptose et l’inhibition de la croissance tumorale. <br><br />
<br />
<br />
En reprenant l’ensemble de ces données, il apparait évident que la mise en application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer du poumon à non petites cellules représente une franche alternative aux traitements déjà existants, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] ciblant l'organe d’intérêt et le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] pouvant délivrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] à l’activité apoptotique confirmée. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Conclusion3Fr#drapeau|Haut de page]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/BiobricksFr#drapeau" target="_self"><br />
<img title="On passe à la page suivante !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Conclusion3FrTeam:SupBiotech-Paris/Conclusion3Fr2009-10-21T17:24:53Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Conclusion =<br />
<br />
<br />
Le cancer du poumon non à petites cellules, ou NSCLC, est un cancer dit agressif. Il se développe dans la lumière de l’organe à une vitesse relativement élevée. Cette localisation tumorale est fortement corrélée à la localisation des facteurs déclenchant la tumorogénèse (contenus dans la fumée du tabac par exemple). Les cellules tumorales perdent leur capacité apoptotique suite à la perte fonctionnelle de divers suppresseurs de tumeur jouant un rôle dans la voie de signalisation de la cascade apoptotique. L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant une version saine des suppresseurs de tumeur non-fonctionnels. Le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] nous a permis de recenser les gènes les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon. Ainsi nous avons pu déterminer la composition du [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] appliqué à cette physiopathologie. <br><br />
<br />
<br />
De nombreuses études ont montrées que le poumon est l'un des tropismes naturels de <i>Mycobactérium avium</i> chez la souris, comme chez l’homme. Après une injection en intraveineuse de <i>M. avium</i>, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] arrivera bien au poumon et pourra s’y développer. Le premier concept du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], pour cette mise en application, est donc validé. Concernant le second, la littérature montre que la création d’un phage recombiné avec [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus, une protéine permettant le passage des membranes par endocytose, est possible. La création du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], qui aura pour objectif d’intégrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] au sein de cellules malades eucaryotes, est donc réalisable. Il est prouvé que l’on peut jouer sur l’efficacité d’interaction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] avec les intégrines des cellules eucaryotes en utilisant soit l’ensemble de sa structure soit son motif RGD seul. Dans le contexte du projet [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], c’est l’ensemble de sa structure que l’on a placé sur la capside du phage Lambda. Possédant alors une moins forte affinité pour les intégrines cellulaires que si on avait utilisé le motif RGD seul, les [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteurs cellulaires]] pourront d’avantage se disperser et par conséquence, toucher un plus grand nombre de cellules.<br><br />
<br />
<br />
Des études <i>in vivo</i> chez l’homme ont prouvées qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui ce gène est muté permet le déclenchement du processus d’apoptose et l’inhibition de la croissance tumorale. <br><br />
<br />
<br />
En reprenant l’ensemble ces données, il apparait évident que la mise en application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer du poumon à non petites cellules représente une franche alternative aux traitements déjà existants, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] ciblant l'organe d’intérêt et le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] pouvant délivrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] à l’activité apoptotique confirmée. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Conclusion3Fr#drapeau|Haut de page]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/BiobricksFr#drapeau" target="_self"><br />
<img title="On passe à la page suivante !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Conclusion3FrTeam:SupBiotech-Paris/Conclusion3Fr2009-10-21T17:21:24Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Conclusion =<br />
<br />
<br />
Le cancer du poumon non à petites cellules, ou NSCLC, est un cancer dit agressif. Il se développe dans la lumière de l’organe à une vitesse relativement élevée. Cette localisation tumorale est fortement corrélée à la localisation des facteurs déclenchant la tumorogénèse (contenus dans la fumée du tabac par exemple). Les cellules tumorales perdent leur capacité apoptotique suite à la perte fonctionnelle de divers suppresseurs de tumeur jouant un role dans la voie de signalisation de la cascade apoptotique. L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant une version saine des suppresseurs de tumeur non-fonctionnels. Le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] nous a permis de recenser les gènes les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon. Ainsi nous avons pu déterminer la composition du [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] appliqué à cette physiopathologie. <br><br />
<br />
<br />
De nombreuses études ont montrées que le poumon est l'un des tropismes naturels de Mycobactérium avium chez la souris, comme chez l’homme. Après une injection en intraveineuse de M. avium, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] arrivera bien au poumon et pourra s’y développer. Le premier concept du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], pour cette mise en application, est donc validé. Concernant le second, la littérature montre que la création d’un phage recombiné avec [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus, une protéine permettant le passage des membranes par endocytose, est possible. La création du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], qui aura pour objectif d’intégrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] au sein de cellules malades eucaryotes, est donc réalisable. Il est prouvé que l’on peut jouer sur l’efficacité d’interaction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] avec les intégrines des cellules eucaryotes en utilisant soit l’ensemble de sa structure soit son motif RGD seul. Dans le contexte du projet [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], c’est l’ensemble de sa structure que l’on a placé sur la capside du phage lambda. Possédant alors une moins forte affinité pour les intégrines cellulaires que si on avait utilisé le motif RGD seul, les [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteurs cellulaires]] pourront d’avantage se disperser et par conséquence, toucher un plus grand nombre de cellules.<br><br />
<br />
<br />
Des études <i>in vivo</i> chez l’homme ont prouvées qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui ce gène est muté permet le déclenchement du processus d’apoptose et l’inhibition de la croissance tumorale. <br><br />
<br />
<br />
En reprenant l’ensemble ces données, il apparait évident que la mise en application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer du poumon à non petites cellules représente une franche alternative aux traitements déjà existants, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] ciblant l'organe d’intérêt et le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] pouvant délivrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] à l’activité apoptotique confirmée. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Conclusion3Fr#drapeau|Haut de page]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/BiobricksFr#drapeau" target="_self"><br />
<img title="On passe à la page suivante !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Conclusion3FrTeam:SupBiotech-Paris/Conclusion3Fr2009-10-21T17:20:49Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
<br />
Le cancer du poumon non à petites cellules, ou NSCLC, est un cancer dit agressif. Il se développe dans la lumière de l’organe à une vitesse relativement élevée. Cette localisation tumorale est fortement corrélée à la localisation des facteurs déclenchant la tumorogénèse (contenus dans la fumée du tabac par exemple). Les cellules tumorales perdent leur capacité apoptotique suite à la perte fonctionnelle de divers suppresseurs de tumeur jouant un role dans la voie de signalisation de la cascade apoptotique. L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant une version saine des suppresseurs de tumeur non-fonctionnels. Le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] nous a permis de recenser les gènes les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon. Ainsi nous avons pu déterminer la composition du [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] appliqué à cette physiopathologie. <br><br />
<br />
<br />
De nombreuses études ont montrées que le poumon est l'un des tropismes naturels de Mycobactérium avium chez la souris, comme chez l’homme. Après une injection en intraveineuse de M. avium, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] arrivera bien au poumon et pourra s’y développer. Le premier concept du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], pour cette mise en application, est donc validé. Concernant le second, la littérature montre que la création d’un phage recombiné avec [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus, une protéine permettant le passage des membranes par endocytose, est possible. La création du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], qui aura pour objectif d’intégrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] au sein de cellules malades eucaryotes, est donc réalisable. Il est prouvé que l’on peut jouer sur l’efficacité d’interaction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] avec les intégrines des cellules eucaryotes en utilisant soit l’ensemble de sa structure soit son motif RGD seul. Dans le contexte du projet [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], c’est l’ensemble de sa structure que l’on a placé sur la capside du phage lambda. Possédant alors une moins forte affinité pour les intégrines cellulaires que si on avait utilisé le motif RGD seul, les [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteurs cellulaires]] pourront d’avantage se disperser et par conséquence, toucher un plus grand nombre de cellules.<br><br />
<br />
<br />
Des études <i>in vivo</i> chez l’homme ont prouvées qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui ce gène est muté permet le déclenchement du processus d’apoptose et l’inhibition de la croissance tumorale. <br><br />
<br />
<br />
En reprenant l’ensemble ces données, il apparait évident que la mise en application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer du poumon à non petites cellules représente une franche alternative aux traitements déjà existants, le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] ciblant l'organe d’intérêt et le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] pouvant délivrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] à l’activité apoptotique confirmée. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Conclusion3Fr#drapeau|Haut de page]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/BiobricksFr#drapeau" target="_self"><br />
<img title="On passe à la page suivante !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/SafetyTeam:SupBiotech-Paris/Safety2009-10-21T15:33:21Z<p>Aurel: /* Judging form */</p>
<hr />
<div>{{Template:Supbiotechcss9.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Safety =<br />
<br />
== Judging form ==<br />
<br />
'''Would any of your project ideas raise safety issues in terms of''': <br><br />
Researcher safety?<br />
:*No, because all hygiene and security rules were followed. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]]) <br><br />
Public safety?<br />
:*No, the DVS is not dangerous for humans. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_pour_la_sant.C3.A9_publique_et_l.27environnement|Explications]])<br><br />
Environmental safety?<br />
:*No, it cannot be spread in the environment ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_pour_la_sant.C3.A9_publique_et_l.27environnement|Explications]] )<br><br />
<br />
'''Is there a local biosafety group, committee, or review board at your institution?''' <br><br />
:*We do not have a department's safety in our school. However there is a committee that is responsible for validating any new project in the laboratory that welcomed us. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]] )<br><br />
<br />
''' What does your local biosafety group think about your project ?''' <br><br />
:*The head of the laboratory agreed to let us develop our project in their premises. Moreover, one of the managers attended our ethical debate ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]])<br><br />
<br />
''' Do any of the new BioBrick parts that you made this year raise any safety issues ?''' <br><br />
:*No, the parts are mostly associations of existing BioBricks. The other Biobrick parts do not involve new safety issues. ([[Team:SupBiotech-Paris/Safety#BioBricks|Explications]] )<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
== Safety for Synthetic Biology ==<br />
<br />
=== Foreword ===<br />
<br />
Synthetic biology is a new approach of biology, which aims to synthesize and design new biological components and systems or redesign existing biological elements in order to create systems performing a specific function. <br><br />
Synthetic biology has grown very rapidly: it has enabled the development of new markets and the reorganization of different actors from biotechnology, energy, and pharmaceuticals to food processing and petrochemicals sectors. Currently, the activities of these companies are separated into two distinct groups: the one of Gene Foundries that synthesize genes and more complex systems on demand, and the one combining the companies developing microorganisms able of producing biofuels, drugs or chemicals.<br><br />
These many applications show the interest of synthetic biology to industry and the various benefits it can bring to society in the future.<br><br />
<br />
However, these advances should not be to the detriment of the safety and security for society. It is therefore necessary to study thoroughly the balance “benefit / risk “ before developing a new use of synthetic biology. Taking into account all these elements is necessary to develop solutions and performing protocols to reassure the society, and thus ensure the sustainability of this new approach of biology.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Introduction ===<br />
<br />
The main concern of the scientific community with regard to synthetic biology is that we cannot really predict the risks. Indeed, current knowledge does not allow us to have hindsight in order to predict the evolution and behavior of synthetic microorganisms. The engineering of biological systems is currently expensive and hazardous mainly because the scientific community does not properly control the molecular and cellular processes to provide reliable solutions.<br><br />
Three different strategies have been identified to address this problem:<br />
:*The first is STANDARDIZATION. It consists in developing and promoting standards which we can also apply the definition, description and characterization of basic biological entities. The creation of the MIT Registry of Standard Biological Parts is a first step in this direction.<br />
:*The second is the DISSOCIATION. This method allows separating a complex problem into a number of simple problems more important. We can thus advance in solving various independent problems, and therefore solve complex systems.<br />
:*The last one is ABSTRACTION. It classifies information that describes the different biological functions, according to a hierarchy (which takes into account different levels of complexity).<br><br />
These different strategies help to normalize and standardize the protocols and methods of synthetic biology in order to ease the use and thus limit the risks.<br />
Pending greater use of these three strategies, it is necessary to anticipate the potential risks for experimentation that will be developed in the near future for the standardization of synthetic biology.<br><br />
Synthetic biology is only in its infancy; it is difficult to determine potential risks. Nevertheless, to discern risk areas, we can transpose the experience gained during the development of recombinant DNA to synthetic biology.<br><br />
We can then distinguish three major types of risks:<br />
:*The first one, microorganisms can escape from their confinement area, proliferate without any control and contaminate the environment, causing damage repairable or not.<br />
:*Secondly, synthetic organisms may develop in an open environment, side effects not detected during testing phases.<br />
:*Finally, it is also important to take into account the risk of bioterrorism. Terrorist organizations or individual actions can exploit synthetic biology for hostile purposes.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Accidental release ===<br />
<br />
It has never yet been mentioned any release of GMOs into the environment. It is likely that GMO is less competitive compared to wild strains and did not persist in the environment.<br />
However, to avoid overflow, the scientific community has developed various means of containment:<br />
:*Physical containment: This is the most used; it ranks bacteria and pathogens agents into 4 classes and determines the requirements to be followed in handling.<br />
:*The trophic containment: It is about making the microorganism dependent of rare substances or unknown in nature, so it cannot grow without human intervention.<br />
:*The containment semantics: It is still in development. It consists for example on modifying the genetic code or the development of new nucleic acids, called XNA which the sugar is neither a desoxyribose or ribose, which prevents the transmission of genes.<br><br />
A final way would be the addition of suicide genes into the genome of the microorganism, destroying the bacteria once performed its function.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Tests in open environment ===<br />
<br />
Since the creation of GMOs and their use in agriculture, many varieties of plants were grown in nature: in an open environment, while the vast majority of species were only tested in the laboratory.<br><br />
In theory, two types of adverse effects may occur after the test of a GMO in an open environment. A fortiori it is the case for Genetically Synthesized organisms (OGS):<br />
:*The microorganism can disturb the local biotope by creating a competitive environment, which may in the worst case lead to the extinction of several species.<br />
:*It may also, after having colonized the middle, becoming impossible to remove.<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
<br />
===Bioterror===<br />
<br />
The rapid development of synthetic biology allows us to create living organisms de novo by enfranchising from the problems of genetic engineering. Many companies have developed to design new genes, segments of DNA and proteins. That popularity has increased access to technology for all. Indeed, sites for bio-yourselfers Sunday "flourish on the Internet, providing knowledge, tips and tricks and also professional equipment.<br><br />
<center>[[Image:CaptureécranEn.png|650px]]recherche ebay</center><br />
<br />
<br />
This personal research conducted on the site [www.ebay.com] demonstrated.<br><br />
Although in most cases the bio-pirates have no intent to defraud, some persons pursue less noble goals:<br><br />
<br />
<center>[[Image:Spectrederisquedebio-terreurEn.png|530px]]</center><br />
<br />
<br />
Many conferences on biosafety have highlighted the need to develop new techniques to reduce the risk of bio-terror.<br />
:*The first is simply to give a sense of responsibility to companies delivering genes by command. They have to verify the recipient of synthesized genes and also the type of genes required (compared with the genomes of known viruses and their toxins). However this cannot be set up in SMEs because it requires great financial resources that reduce the net profits of the company.<br />
:*The second is the research of defense strategies to fight against bio-terrorism.<br />
:*A final technique would also list all machines that can help to synthesize new genes, even if it is costly in time and financial resources. <br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
===Conclusion===<br />
<br />
In society today, many people are favorable to reduce and regulate the practice of synthetic biology until all risks are identified and excluded in accordance with the principle of precaution. However the application of this principle highlights a paradox, which harms innovation:<br><br />
To use the techniques of synthetic biology, all risks must be known and controlled. But these risks cannot be determined if we do not progress by practicing synthetic biology. This is what the precautionary principle refuses because synthetic biology is not yet reliable.<br><br />
[[Image:PrincipedeprécautionEn.jpg|center]]<br><br />
The precautionary principle refuses innovation, as long as there is the slightest risk.<br><br />
Conversely, the principle of responsibility can focus its work on the most serious risk after having considered all the risks. It is a conceivable solution to establish regulations; it allows pursuing researches despite uncontrolled risks.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==Safety in DVS project==<br />
<br />
===Foreword===<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
===Safety for society and the environment===<br />
<br />
==== ''Mycobacterium avium'' subtype ''avium'' ====<br />
<br />
<br />
''Mycobacterium avium'' subtype ''avium'' is rarely responsible for serious infections in humans. As Willy Rozenbaum (co discoverer of HIV) said at our ethical debate, "is a bacterium that is very ubiquitous, it is found in faucet water, we are almost all contaminated.<br><br />
<br />
''Mycobacterium avium'' is "safe" for the environment and also for healthy people. Indeed, if there is an infection, it is localized in the organism. Conversely, infection is more global for immunodepressed patients (due to HIV for example). The effects of infection are, of course, to analyze in different phenotypes (healthy, tumor, immunodepressed) to investigate the benefit / risk balance for the patient. <br><br />
<br />
Moreover, our ''Mycobacterium'' does not subsist in the organism. It is indeed lysed during the liberation of phage in the tissue after the addition of doxycycline. <br><br />
<br />
<br />
Regarding doxycycline, it has side effects such as staining of bones, toxic epidermal necrolysis, the Stevens Johnson syndrome, megaloblastic anemia, esophageal ulceration, neuromuscular block, cutaneous porphyria, or dyschromatopsia. However, this antibiotic is already widely used by the pharmaceutical industry and does not present any danger as long as the doses and contraindication are respected. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
====Lambda Phage====<br />
<br />
<br />
Our cell vector encapsidated only the therapeutic plasmid (not the entire genome). If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid. <br><br />
<br />
Moreover, our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world". <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==== Therapeutic Plasmid ====<br />
<br />
<br />
The therapeutic plasmid brings to the organism tumor suppressor genes (p53, p16, ATM, RB1 or PTEN) wild type. The promoters added are those present in healthy cells: so there is no increased synthesis of tumor suppressor genes in healthy cells. The regulation is not modified. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==== BioBricks ====<br />
<br />
<br />
Our biobricks set no safety problems : <br />
*The biobrick for COS sequence may set a security problem since it allows the encapsidation of any sequence within the bacteriophage lambda. We can therefore encapsidate “harmful” sequences in a recombinant phage. But this biobrick was not subjected to iGEM. <br><br />
*Some of our biobricks are an assembling of bricks already existing in the register of MIT. <br><br />
*For other biobricks, it is a DNA targeting sequence (DTS), which recruits nuclear localization sequence, a factor specific stem cells (CFS) and a capsid protein of phage lambda synthesized from its own genome. Thus it does not cover new issues of safety. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Laboratory safety===<br />
<br />
<br />
We do not have a department's safety in our school. However in the laboratory that welcomed us, there is a committee that is responsible for validating any new project. <br><br />
<br />
During various experiments, a strict physical containment of our microorganisms was achieved and all the rules in the laboratory were followed. (Fume hood for handling dangerous substances, sterile containers, sorting waste, contaminated waste discharged into biological bins...) <br><br />
<br />
Good laboratory practices for the experiments on mice were also followed. (Renewal of litter, respect of the animal, reducing the number of animals used...) <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Gallery#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/SafetyTeam:SupBiotech-Paris/Safety2009-10-21T15:29:33Z<p>Aurel: /* Laboratory safety */</p>
<hr />
<div>{{Template:Supbiotechcss9.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Safety =<br />
<br />
== Judging form ==<br />
<br />
'''Would any of your project ideas raise safety issues in terms of’’’: <br><br />
Researcher safety?<br />
:*No, because all hygiene and security rules were followed. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]]) <br><br />
Public safety?<br />
:*No, the DVS is not dangerous for humans. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_pour_la_sant.C3.A9_publique_et_l.27environnement|Explications]])<br><br />
Environmental safety?<br />
:*No, it cannot be spread in the environment ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_pour_la_sant.C3.A9_publique_et_l.27environnement|Explications]] )<br><br />
<br />
'''Is there a local biosafety group, committee, or review board at your institution?''' <br><br />
:*We do not have a department's safety in our school. However there is a committee that is responsible for validating any new project in the laboratory that welcomed us. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]] )<br><br />
<br />
''' What does your local biosafety group think about your project ?''' <br><br />
:*The head of the laboratory agreed to let us develop our project in their premises. Moreover, one of the managers attended our ethical debate ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]])<br><br />
<br />
''' Do any of the new BioBrick parts that you made this year raise any safety issues ?''' <br><br />
:*No, the parts are mostly associations of existing BioBricks. The other Biobrick parts do not involve new safety issues. ([[Team:SupBiotech-Paris/Safety#BioBricks|Explications]] )<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
== Safety for Synthetic Biology ==<br />
<br />
=== Foreword ===<br />
<br />
Synthetic biology is a new approach of biology, which aims to synthesize and design new biological components and systems or redesign existing biological elements in order to create systems performing a specific function. <br><br />
Synthetic biology has grown very rapidly: it has enabled the development of new markets and the reorganization of different actors from biotechnology, energy, and pharmaceuticals to food processing and petrochemicals sectors. Currently, the activities of these companies are separated into two distinct groups: the one of Gene Foundries that synthesize genes and more complex systems on demand, and the one combining the companies developing microorganisms able of producing biofuels, drugs or chemicals.<br><br />
These many applications show the interest of synthetic biology to industry and the various benefits it can bring to society in the future.<br><br />
<br />
However, these advances should not be to the detriment of the safety and security for society. It is therefore necessary to study thoroughly the balance “benefit / risk “ before developing a new use of synthetic biology. Taking into account all these elements is necessary to develop solutions and performing protocols to reassure the society, and thus ensure the sustainability of this new approach of biology.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Introduction ===<br />
<br />
The main concern of the scientific community with regard to synthetic biology is that we cannot really predict the risks. Indeed, current knowledge does not allow us to have hindsight in order to predict the evolution and behavior of synthetic microorganisms. The engineering of biological systems is currently expensive and hazardous mainly because the scientific community does not properly control the molecular and cellular processes to provide reliable solutions.<br><br />
Three different strategies have been identified to address this problem:<br />
:*The first is STANDARDIZATION. It consists in developing and promoting standards which we can also apply the definition, description and characterization of basic biological entities. The creation of the MIT Registry of Standard Biological Parts is a first step in this direction.<br />
:*The second is the DISSOCIATION. This method allows separating a complex problem into a number of simple problems more important. We can thus advance in solving various independent problems, and therefore solve complex systems.<br />
:*The last one is ABSTRACTION. It classifies information that describes the different biological functions, according to a hierarchy (which takes into account different levels of complexity).<br><br />
These different strategies help to normalize and standardize the protocols and methods of synthetic biology in order to ease the use and thus limit the risks.<br />
Pending greater use of these three strategies, it is necessary to anticipate the potential risks for experimentation that will be developed in the near future for the standardization of synthetic biology.<br><br />
Synthetic biology is only in its infancy; it is difficult to determine potential risks. Nevertheless, to discern risk areas, we can transpose the experience gained during the development of recombinant DNA to synthetic biology.<br><br />
We can then distinguish three major types of risks:<br />
:*The first one, microorganisms can escape from their confinement area, proliferate without any control and contaminate the environment, causing damage repairable or not.<br />
:*Secondly, synthetic organisms may develop in an open environment, side effects not detected during testing phases.<br />
:*Finally, it is also important to take into account the risk of bioterrorism. Terrorist organizations or individual actions can exploit synthetic biology for hostile purposes.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Accidental release ===<br />
<br />
It has never yet been mentioned any release of GMOs into the environment. It is likely that GMO is less competitive compared to wild strains and did not persist in the environment.<br />
However, to avoid overflow, the scientific community has developed various means of containment:<br />
:*Physical containment: This is the most used; it ranks bacteria and pathogens agents into 4 classes and determines the requirements to be followed in handling.<br />
:*The trophic containment: It is about making the microorganism dependent of rare substances or unknown in nature, so it cannot grow without human intervention.<br />
:*The containment semantics: It is still in development. It consists for example on modifying the genetic code or the development of new nucleic acids, called XNA which the sugar is neither a desoxyribose or ribose, which prevents the transmission of genes.<br><br />
A final way would be the addition of suicide genes into the genome of the microorganism, destroying the bacteria once performed its function.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Tests in open environment ===<br />
<br />
Since the creation of GMOs and their use in agriculture, many varieties of plants were grown in nature: in an open environment, while the vast majority of species were only tested in the laboratory.<br><br />
In theory, two types of adverse effects may occur after the test of a GMO in an open environment. A fortiori it is the case for Genetically Synthesized organisms (OGS):<br />
:*The microorganism can disturb the local biotope by creating a competitive environment, which may in the worst case lead to the extinction of several species.<br />
:*It may also, after having colonized the middle, becoming impossible to remove.<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
<br />
===Bioterror===<br />
<br />
The rapid development of synthetic biology allows us to create living organisms de novo by enfranchising from the problems of genetic engineering. Many companies have developed to design new genes, segments of DNA and proteins. That popularity has increased access to technology for all. Indeed, sites for bio-yourselfers Sunday "flourish on the Internet, providing knowledge, tips and tricks and also professional equipment.<br><br />
<center>[[Image:CaptureécranEn.png|650px]]recherche ebay</center><br />
<br />
<br />
This personal research conducted on the site [www.ebay.com] demonstrated.<br><br />
Although in most cases the bio-pirates have no intent to defraud, some persons pursue less noble goals:<br><br />
<br />
<center>[[Image:Spectrederisquedebio-terreurEn.png|530px]]</center><br />
<br />
<br />
Many conferences on biosafety have highlighted the need to develop new techniques to reduce the risk of bio-terror.<br />
:*The first is simply to give a sense of responsibility to companies delivering genes by command. They have to verify the recipient of synthesized genes and also the type of genes required (compared with the genomes of known viruses and their toxins). However this cannot be set up in SMEs because it requires great financial resources that reduce the net profits of the company.<br />
:*The second is the research of defense strategies to fight against bio-terrorism.<br />
:*A final technique would also list all machines that can help to synthesize new genes, even if it is costly in time and financial resources. <br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
===Conclusion===<br />
<br />
In society today, many people are favorable to reduce and regulate the practice of synthetic biology until all risks are identified and excluded in accordance with the principle of precaution. However the application of this principle highlights a paradox, which harms innovation:<br><br />
To use the techniques of synthetic biology, all risks must be known and controlled. But these risks cannot be determined if we do not progress by practicing synthetic biology. This is what the precautionary principle refuses because synthetic biology is not yet reliable.<br><br />
[[Image:PrincipedeprécautionEn.jpg|center]]<br><br />
The precautionary principle refuses innovation, as long as there is the slightest risk.<br><br />
Conversely, the principle of responsibility can focus its work on the most serious risk after having considered all the risks. It is a conceivable solution to establish regulations; it allows pursuing researches despite uncontrolled risks.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==Safety in DVS project==<br />
<br />
===Foreword===<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
===Safety for society and the environment===<br />
<br />
==== ''Mycobacterium avium'' subtype ''avium'' ====<br />
<br />
<br />
''Mycobacterium avium'' subtype ''avium'' is rarely responsible for serious infections in humans. As Willy Rozenbaum (co discoverer of HIV) said at our ethical debate, "is a bacterium that is very ubiquitous, it is found in faucet water, we are almost all contaminated.<br><br />
<br />
''Mycobacterium avium'' is "safe" for the environment and also for healthy people. Indeed, if there is an infection, it is localized in the organism. Conversely, infection is more global for immunodepressed patients (due to HIV for example). The effects of infection are, of course, to analyze in different phenotypes (healthy, tumor, immunodepressed) to investigate the benefit / risk balance for the patient. <br><br />
<br />
Moreover, our ''Mycobacterium'' does not subsist in the organism. It is indeed lysed during the liberation of phage in the tissue after the addition of doxycycline. <br><br />
<br />
<br />
Regarding doxycycline, it has side effects such as staining of bones, toxic epidermal necrolysis, the Stevens Johnson syndrome, megaloblastic anemia, esophageal ulceration, neuromuscular block, cutaneous porphyria, or dyschromatopsia. However, this antibiotic is already widely used by the pharmaceutical industry and does not present any danger as long as the doses and contraindication are respected. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
====Lambda Phage====<br />
<br />
<br />
Our cell vector encapsidated only the therapeutic plasmid (not the entire genome). If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid. <br><br />
<br />
Moreover, our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world". <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==== Therapeutic Plasmid ====<br />
<br />
<br />
The therapeutic plasmid brings to the organism tumor suppressor genes (p53, p16, ATM, RB1 or PTEN) wild type. The promoters added are those present in healthy cells: so there is no increased synthesis of tumor suppressor genes in healthy cells. The regulation is not modified. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==== BioBricks ====<br />
<br />
<br />
Our biobricks set no safety problems : <br />
*The biobrick for COS sequence may set a security problem since it allows the encapsidation of any sequence within the bacteriophage lambda. We can therefore encapsidate “harmful” sequences in a recombinant phage. But this biobrick was not subjected to iGEM. <br><br />
*Some of our biobricks are an assembling of bricks already existing in the register of MIT. <br><br />
*For other biobricks, it is a DNA targeting sequence (DTS), which recruits nuclear localization sequence, a factor specific stem cells (CFS) and a capsid protein of phage lambda synthesized from its own genome. Thus it does not cover new issues of safety. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Laboratory safety===<br />
<br />
<br />
We do not have a department's safety in our school. However in the laboratory that welcomed us, there is a committee that is responsible for validating any new project. <br><br />
<br />
During various experiments, a strict physical containment of our microorganisms was achieved and all the rules in the laboratory were followed. (Fume hood for handling dangerous substances, sterile containers, sorting waste, contaminated waste discharged into biological bins...) <br><br />
<br />
Good laboratory practices for the experiments on mice were also followed. (Renewal of litter, respect of the animal, reducing the number of animals used...) <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Gallery#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/SafetyTeam:SupBiotech-Paris/Safety2009-10-21T15:29:09Z<p>Aurel: /* Laboratory safety */</p>
<hr />
<div>{{Template:Supbiotechcss9.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Safety =<br />
<br />
== Judging form ==<br />
<br />
'''Would any of your project ideas raise safety issues in terms of’’’: <br><br />
Researcher safety?<br />
:*No, because all hygiene and security rules were followed. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]]) <br><br />
Public safety?<br />
:*No, the DVS is not dangerous for humans. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_pour_la_sant.C3.A9_publique_et_l.27environnement|Explications]])<br><br />
Environmental safety?<br />
:*No, it cannot be spread in the environment ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_pour_la_sant.C3.A9_publique_et_l.27environnement|Explications]] )<br><br />
<br />
'''Is there a local biosafety group, committee, or review board at your institution?''' <br><br />
:*We do not have a department's safety in our school. However there is a committee that is responsible for validating any new project in the laboratory that welcomed us. ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]] )<br><br />
<br />
''' What does your local biosafety group think about your project ?''' <br><br />
:*The head of the laboratory agreed to let us develop our project in their premises. Moreover, one of the managers attended our ethical debate ([[Team:SupBiotech-Paris/Safety#S.C3.BBret.C3.A9_en_laboratoire|Explications]])<br><br />
<br />
''' Do any of the new BioBrick parts that you made this year raise any safety issues ?''' <br><br />
:*No, the parts are mostly associations of existing BioBricks. The other Biobrick parts do not involve new safety issues. ([[Team:SupBiotech-Paris/Safety#BioBricks|Explications]] )<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
== Safety for Synthetic Biology ==<br />
<br />
=== Foreword ===<br />
<br />
Synthetic biology is a new approach of biology, which aims to synthesize and design new biological components and systems or redesign existing biological elements in order to create systems performing a specific function. <br><br />
Synthetic biology has grown very rapidly: it has enabled the development of new markets and the reorganization of different actors from biotechnology, energy, and pharmaceuticals to food processing and petrochemicals sectors. Currently, the activities of these companies are separated into two distinct groups: the one of Gene Foundries that synthesize genes and more complex systems on demand, and the one combining the companies developing microorganisms able of producing biofuels, drugs or chemicals.<br><br />
These many applications show the interest of synthetic biology to industry and the various benefits it can bring to society in the future.<br><br />
<br />
However, these advances should not be to the detriment of the safety and security for society. It is therefore necessary to study thoroughly the balance “benefit / risk “ before developing a new use of synthetic biology. Taking into account all these elements is necessary to develop solutions and performing protocols to reassure the society, and thus ensure the sustainability of this new approach of biology.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Introduction ===<br />
<br />
The main concern of the scientific community with regard to synthetic biology is that we cannot really predict the risks. Indeed, current knowledge does not allow us to have hindsight in order to predict the evolution and behavior of synthetic microorganisms. The engineering of biological systems is currently expensive and hazardous mainly because the scientific community does not properly control the molecular and cellular processes to provide reliable solutions.<br><br />
Three different strategies have been identified to address this problem:<br />
:*The first is STANDARDIZATION. It consists in developing and promoting standards which we can also apply the definition, description and characterization of basic biological entities. The creation of the MIT Registry of Standard Biological Parts is a first step in this direction.<br />
:*The second is the DISSOCIATION. This method allows separating a complex problem into a number of simple problems more important. We can thus advance in solving various independent problems, and therefore solve complex systems.<br />
:*The last one is ABSTRACTION. It classifies information that describes the different biological functions, according to a hierarchy (which takes into account different levels of complexity).<br><br />
These different strategies help to normalize and standardize the protocols and methods of synthetic biology in order to ease the use and thus limit the risks.<br />
Pending greater use of these three strategies, it is necessary to anticipate the potential risks for experimentation that will be developed in the near future for the standardization of synthetic biology.<br><br />
Synthetic biology is only in its infancy; it is difficult to determine potential risks. Nevertheless, to discern risk areas, we can transpose the experience gained during the development of recombinant DNA to synthetic biology.<br><br />
We can then distinguish three major types of risks:<br />
:*The first one, microorganisms can escape from their confinement area, proliferate without any control and contaminate the environment, causing damage repairable or not.<br />
:*Secondly, synthetic organisms may develop in an open environment, side effects not detected during testing phases.<br />
:*Finally, it is also important to take into account the risk of bioterrorism. Terrorist organizations or individual actions can exploit synthetic biology for hostile purposes.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Accidental release ===<br />
<br />
It has never yet been mentioned any release of GMOs into the environment. It is likely that GMO is less competitive compared to wild strains and did not persist in the environment.<br />
However, to avoid overflow, the scientific community has developed various means of containment:<br />
:*Physical containment: This is the most used; it ranks bacteria and pathogens agents into 4 classes and determines the requirements to be followed in handling.<br />
:*The trophic containment: It is about making the microorganism dependent of rare substances or unknown in nature, so it cannot grow without human intervention.<br />
:*The containment semantics: It is still in development. It consists for example on modifying the genetic code or the development of new nucleic acids, called XNA which the sugar is neither a desoxyribose or ribose, which prevents the transmission of genes.<br><br />
A final way would be the addition of suicide genes into the genome of the microorganism, destroying the bacteria once performed its function.<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Tests in open environment ===<br />
<br />
Since the creation of GMOs and their use in agriculture, many varieties of plants were grown in nature: in an open environment, while the vast majority of species were only tested in the laboratory.<br><br />
In theory, two types of adverse effects may occur after the test of a GMO in an open environment. A fortiori it is the case for Genetically Synthesized organisms (OGS):<br />
:*The microorganism can disturb the local biotope by creating a competitive environment, which may in the worst case lead to the extinction of several species.<br />
:*It may also, after having colonized the middle, becoming impossible to remove.<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
<br />
===Bioterror===<br />
<br />
The rapid development of synthetic biology allows us to create living organisms de novo by enfranchising from the problems of genetic engineering. Many companies have developed to design new genes, segments of DNA and proteins. That popularity has increased access to technology for all. Indeed, sites for bio-yourselfers Sunday "flourish on the Internet, providing knowledge, tips and tricks and also professional equipment.<br><br />
<center>[[Image:CaptureécranEn.png|650px]]recherche ebay</center><br />
<br />
<br />
This personal research conducted on the site [www.ebay.com] demonstrated.<br><br />
Although in most cases the bio-pirates have no intent to defraud, some persons pursue less noble goals:<br><br />
<br />
<center>[[Image:Spectrederisquedebio-terreurEn.png|530px]]</center><br />
<br />
<br />
Many conferences on biosafety have highlighted the need to develop new techniques to reduce the risk of bio-terror.<br />
:*The first is simply to give a sense of responsibility to companies delivering genes by command. They have to verify the recipient of synthesized genes and also the type of genes required (compared with the genomes of known viruses and their toxins). However this cannot be set up in SMEs because it requires great financial resources that reduce the net profits of the company.<br />
:*The second is the research of defense strategies to fight against bio-terrorism.<br />
:*A final technique would also list all machines that can help to synthesize new genes, even if it is costly in time and financial resources. <br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
===Conclusion===<br />
<br />
In society today, many people are favorable to reduce and regulate the practice of synthetic biology until all risks are identified and excluded in accordance with the principle of precaution. However the application of this principle highlights a paradox, which harms innovation:<br><br />
To use the techniques of synthetic biology, all risks must be known and controlled. But these risks cannot be determined if we do not progress by practicing synthetic biology. This is what the precautionary principle refuses because synthetic biology is not yet reliable.<br><br />
[[Image:PrincipedeprécautionEn.jpg|center]]<br><br />
The precautionary principle refuses innovation, as long as there is the slightest risk.<br><br />
Conversely, the principle of responsibility can focus its work on the most serious risk after having considered all the risks. It is a conceivable solution to establish regulations; it allows pursuing researches despite uncontrolled risks.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==Safety in DVS project==<br />
<br />
===Foreword===<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
===Safety for society and the environment===<br />
<br />
==== ''Mycobacterium avium'' subtype ''avium'' ====<br />
<br />
<br />
''Mycobacterium avium'' subtype ''avium'' is rarely responsible for serious infections in humans. As Willy Rozenbaum (co discoverer of HIV) said at our ethical debate, "is a bacterium that is very ubiquitous, it is found in faucet water, we are almost all contaminated.<br><br />
<br />
''Mycobacterium avium'' is "safe" for the environment and also for healthy people. Indeed, if there is an infection, it is localized in the organism. Conversely, infection is more global for immunodepressed patients (due to HIV for example). The effects of infection are, of course, to analyze in different phenotypes (healthy, tumor, immunodepressed) to investigate the benefit / risk balance for the patient. <br><br />
<br />
Moreover, our ''Mycobacterium'' does not subsist in the organism. It is indeed lysed during the liberation of phage in the tissue after the addition of doxycycline. <br><br />
<br />
<br />
Regarding doxycycline, it has side effects such as staining of bones, toxic epidermal necrolysis, the Stevens Johnson syndrome, megaloblastic anemia, esophageal ulceration, neuromuscular block, cutaneous porphyria, or dyschromatopsia. However, this antibiotic is already widely used by the pharmaceutical industry and does not present any danger as long as the doses and contraindication are respected. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
====Lambda Phage====<br />
<br />
<br />
Our cell vector encapsidated only the therapeutic plasmid (not the entire genome). If it infects bacteria of the commensal flora of the organism (which may be limited by changes in protein internalization), the bacteria will receive just the therapeutic plasmid. <br><br />
<br />
Moreover, our phage is a prokaryote, but cells of human body are eukaryotes. It can therefore be no risk of homologous recombination or integration between its genome and our cells genome, as they do not belong to the same "world". <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==== Therapeutic Plasmid ====<br />
<br />
<br />
The therapeutic plasmid brings to the organism tumor suppressor genes (p53, p16, ATM, RB1 or PTEN) wild type. The promoters added are those present in healthy cells: so there is no increased synthesis of tumor suppressor genes in healthy cells. The regulation is not modified. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
==== BioBricks ====<br />
<br />
<br />
Our biobricks set no safety problems : <br />
*The biobrick for COS sequence may set a security problem since it allows the encapsidation of any sequence within the bacteriophage lambda. We can therefore encapsidate “harmful” sequences in a recombinant phage. But this biobrick was not subjected to iGEM. <br><br />
*Some of our biobricks are an assembling of bricks already existing in the register of MIT. <br><br />
*For other biobricks, it is a DNA targeting sequence (DTS), which recruits nuclear localization sequence, a factor specific stem cells (CFS) and a capsid protein of phage lambda synthesized from its own genome. Thus it does not cover new issues of safety. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
=== Laboratory safety===<br />
<br />
<br />
We do not have a department's safety in our school. However in the laboratory that welcomed us, there is a committee that is responsible for validating any new project. <br><br />
<br />
During various experiments, a strict physical containment of our microorganisms was achieved and all the rules in the laboratory were followed. (Fume hood for handling dangerous substances, sterile containers, sorting waste, contaminated waste discharged into biological bins...) <br><br />
<br />
Good laboratory practices for the experiments on mice were also followed. (Renewal of litter, respect of the animal, reducing the number of animals used...) <br><br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Safety#drapeau|Back to top]]</span><br />
<br />
<br />
<html><br />
<div style="float: right; margin-right: -85px;"><br />
<a href="https://2009.igem.org/Team:SupBiotech-Paris/Gallery#drapeau" target="_self"><br />
<img title="Let's go to the next page !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
</a></div><br />
</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T14:39:06Z<p>Aurel: /* Design of the fusion protein */</p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 11) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
<br />
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<br />
<br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T14:38:00Z<p>Aurel: /* Design de la protéine de fusion */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens celle du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]]. Ce dernier est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]], acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]]. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3' <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] = 1715bp pour l’échantillon 11 et la de protéine D = 385bp pour l’échantillon 7 et 8. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (7 et 8) et de la base de penton (9, 10, 11) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (LambdaPb286-393), des phages lambda recombinés avec la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes. Cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (fragment 1-571) car l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée des bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
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La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
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En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats [1,2] ==<br />
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Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
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<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
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<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
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<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
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<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
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<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusion [3,4,5,6,7,8,9] == <br />
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Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
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La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T14:36:40Z<p>Aurel: /* Design of the fusion protein */</p>
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<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
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= Cell targeting =<br />
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== Context ==<br />
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After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
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==Objective ==<br />
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Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
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== Experimental approach ==<br />
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In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
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Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
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== Results ==<br />
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=== Design of the fusion protein ===<br />
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For the fusion protein design, we decided to extract separately the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
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First and second pair’s genes extraction: <br><br />
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D protein of the Lambda phage: <br><br />
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Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
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Adenovirus 5 penton base : <br><br />
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Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
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Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
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Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3'<br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 7 and 8). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (7 and 8) and penton base (9, 10, 11) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
<br />
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<br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T14:34:56Z<p>Aurel: /* Design de la protéine de fusion */</p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens celle du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]]. Ce dernier est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]], acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]]. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3' <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (7 et 8) et de la base de penton (9, 10, 11) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (LambdaPb286-393), des phages lambda recombinés avec la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes. Cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (fragment 1-571) car l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée des bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
<br />
C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
<br />
[[image: gènes mutés.jpeg|center]]<br />
<br />
Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== L’objectif ==<br />
<br />
L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
<br />
== Démarche expérimentale ==<br />
<br />
<br />
=== Lignée cancéreuse et gène apporté ===<br />
<br />
Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Incorporation du gène TP53 ===<br />
<br />
L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
<br />
<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T14:22:11Z<p>Aurel: /* Contexte */</p>
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= Le Ciblage cellulaire =<br />
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== Contexte ==<br />
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Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens celle du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]]. Ce dernier est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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==Objectif ==<br />
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Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Démarche expérimentale ==<br />
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Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
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Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]], acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
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=== Design de la protéine de fusion ===<br />
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Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]]. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : 5' ATG-ACG-AGC-AAA-GAA-ACC-TT 3'; <br><br />
Reverse : 5' AAA-AAA-ATC-CCG-TAA-AAA-AAG-C 3'. <br><br />
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Base de penton de l’adénovirus 5 : <br><br />
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Forward : 5' AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G 3' <br><br />
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Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
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Forward : 5' CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T 3' <br><br />
Reverse : 5' AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC 3' <br><br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
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Forward : 5' CTT-AAG-CGC-CGG-CGA-AGA-TC 3' <br><br />
Reverse : 5' CTG-CAG-CGG-CCG-CTA-CTA-GTA 3' <br><br><br />
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Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
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Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (LambdaPb286-393), des phages lambda recombinés avec la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Discussion ==<br />
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Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusions ==<br />
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Pour conclure le fragment RGD seul de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes. Cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (fragment 1-571) car l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée des bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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= Le Plasmide antitumoral =<br />
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== Contexte ==<br />
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Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
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<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
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La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
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En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats [1,2] ==<br />
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Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
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<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
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<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
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<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
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<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
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<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusion [3,4,5,6,7,8,9] == <br />
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Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
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La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T11:43:48Z<p>Aurel: </p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
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= Le Ciblage cellulaire =<br />
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== Contexte ==<br />
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Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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==Objectif ==<br />
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Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Démarche expérimentale ==<br />
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Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
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Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]], acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
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=== Design de la protéine de fusion ===<br />
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Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]]. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
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Base de penton de l’adénovirus 5 : <br><br />
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Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
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Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
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Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
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Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (LambdaPb286-393), des phages lambda recombinés avec la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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== Discussion ==<br />
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Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusions ==<br />
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Pour conclure le fragment RGD seul de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes. Cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] (fragment 1-571) car l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée des bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la [[Team:SupBiotech-Paris/Concept2Fr#PB|base de penton]] est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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= Le Plasmide antitumoral =<br />
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== Contexte ==<br />
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Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
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<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
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<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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<br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
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<br />
<br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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<br />
<br />
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<br />
<br />
<br />
<br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Concept2FrTeam:SupBiotech-Paris/Concept2Fr2009-10-21T11:39:19Z<p>Aurel: /* L’internalisation du vecteur cellulaire et la libération du plasmide thérapeutique */</p>
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<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisFr}}<br />
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= Vecteur cellulaire =<br />
<br />
Le vecteur cellulaire a pour fonction d’atteindre la cellule cible et de transfecter [[Team:SupBiotech-Paris/Concept3Fr#drapeau|l’insert génique thérapeutique]].<br><br />
Il doit posséder certaines caractéristiques pour être un vecteur de ciblage cellulaire efficace. Il doit cibler spécifiquement le type cellulaire d’intérêt, celui à soigner. En tant que vecteur de type phagique, il doit être capable de passer la membrane de la cellule eucaryote et y délivrer son contenu de manière efficace.<br><br />
<br><br />
Vous allez pouvoir découvrir au cours de ce chapitre sur quelles caractéristiques nous nous sommes basés pour choisir le '''meilleur bactériophage'''.<br><br />
Puis, vous découvrirez plus en détails ses propriétés au travers des différentes contraintes imposées par le [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] concernant sa '''[[Team:SupBiotech-Paris/Concept1Fr#DT|délivrance tissulaire]]''', '''l’encapsidation du [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]]''' et le '''ciblage cellulaire'''.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Concept2Fr#drapeau|Haut de page]]</span><br />
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== Le vecteur cellulaire : Le bactériophage Lambda [1,2] ==<br />
<br />
Nous avons étudié plusieurs types de bactériophages afin de sélectionner le vecteur cellulaire le plus modulable et le plus contrôlable. Pour se faire, nous avons répertorié tous les types de phages et au sein de chaque type, les espèces les mieux décrites. Plusieurs candidats ont retenu notre attention, mais c’est le bactériophage lambda qui nous a semblé le plus adapté.<br><br />
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Le bactériophage lambda est un virus procaryote qui infecte naturellement la bactérie Escherichia Coli. C’est un bactériophage dit tempéré, car il peut alterner phase lytique et phase lysogène sous certaines conditions. Pendant le cycle lytique, l’ADN circulaire est répliqué en grande quantité dans le cytoplasme, des progénies viraux sont formés, conduisant à la mort de la bactérie et à la libération des virions. Au cours du cycle lysogène, le génome du bactériophage est intégré dans le chromosome bactérien et est transmis à la descendance. La réplication se fait alors au rythme des divisions de la bactérie.<br><br />
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Naturellement infectieux pour la bactérie Escherichia Coli, le bactériophage lambda, comme tous phages, n’est pas infectieux pour l’Homme. Cette caractéristique provient du fait que leur génome est régie par des promoteurs procaryotes mais également du fait qu’ils aient une très faible capacité de transfection des cellules eucaryotes. <br><br />
Enfin, l’une des caractéristiques intéressantes du bactériophage lambda est qu’il possède un ADN double brin linéaire ayant des extrémités cohésives : 12 nucléotides simple brin complémentaire, appelés séquences « Cos ». Ces extrémités cohésives ont une fonction dans '''l’encapsidation génomique'''.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Concept2Fr#drapeau|Haut de page]]</span><br />
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== L’encapsidation du plasmide thérapeutique [2,3,4,5,6,7] ==<br />
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L’encapsidation du génome phagique est particulière chez le bactériophage lambda. Elle est guidée par la [[Team:SupBiotech-Paris/Concept3Fr#cos|séquence Cos]]. Cette caractéristique permet l’encapsidation spécifique de séquence d’intérêt.<br><br />
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[[Image:Encapsidation.png|center|650px|frameless]]<br />
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On observe sur le schéma ci-dessus que l’encapsidation du génome commence et se termine par les [[Team:SupBiotech-Paris/Concept3Fr#cos|séquences Cos]]. Tout ce qui se trouve entre ces séquences est encapsidé. Cette particularité est utilisée en biologie moléculaire depuis de nombreuses années pour la création de banque d’ADN et d’outils moléculaires nommés Cosmides.<br><br />
Nous sommes partis de ce constat pour encapsider, à l’intérieur du vecteur cellulaire, uniquement le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]]. Pour se faire le génome du vecteur, le bactériophage lambda, est délété de ses [[Team:SupBiotech-Paris/Concept3Fr#cos|séquences Cos]], ces dernières sont placées sur le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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Séquence non codante cos : Position 1-132 et 48462-48502 : 174 paires de bases.<br><br />
1.....TATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTAC<br><br />
41....GGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATT<br><br />
81....TTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTT<br><br />
121...AATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAAC<br><br />
161...GACAGGTGCTGAA<br><br />
<br />
[[Image :Schémades2plasmides|center|frameless]]<br />
Schéma des deux plasmides : génome de lambda recombiné + plasmide d’intérêt<br />
<br />
Le vecteur cellulaire encapside le plasmide thérapeutique par l’intermédiaire des [[Team:SupBiotech-Paris/Concept3Fr#cos|séquences Cos]]. Ainsi, il ne délivre que l’[[Team:SupBiotech-Paris/Concept3Fr#drapeau|agent thérapeutique]] dans les cellules, ce qui permet une précaution supplémentaire au cas où le vecteur d’intérêt ne serait pas dans l’organe cible.<br><br />
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== La délivrance du vecteur cellulaire dans l’organe cible [1,2,8]==<br />
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Le vecteur cellulaire possède deux phases d’action, une phase de '''latence''' et une phase d’'''expression et de libération'''. Lors de la phase de latence, appelée '''cycle lysogène''', le génome reste « inactif » et aucun vecteur cellulaire n’est produit au sein du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]]. Alors que pendant la phase d’expression et de libération, appelée '''cycle lytique''', le génome est transcrit et répliqué, afin de produire des vecteurs cellulaires.<br><br />
Cependant le vecteur tissulaire n’est pas l’hôte naturelle du vecteur cellulaire. Ainsi, si l’on veut induire une phase de latence, il nous faut la provoquer. Le système de délivrance implanté dans le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] induit justement cette phase de latence.<br><br />
Le plasmide de contrôle de délivrance du vecteur cellulaire induit un cycle lysogène chez ce dernier. <br><br />
Le cycle lysogène est un phénomène naturellement présent chez le bactériophage lambda, le vecteur cellulaire. Lors de ce cycle, le génome phagique est inséré dans le génome bactérien sous la forme d’un prophage, il devient partie intégrante du génome de l’hôte.<br><br />
A l’état de prophage, toute transcription est réprimée. Seul le gène cI, responsable du maintien du cycle lysogène, est exprimé.<br><br />
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La fixation de cI sur les opérateurs de la région de contrôle des promoteurs pR et pL, bloque l’expression des gènes et permet le maintien de la lysogénie. cI bloque de surcroit son propre promoteur, pRM, permettant ainsi une autorégulation de sa transcription.<br><br />
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Le bactériophage est maintenu sous la forme lysogène aussi longtemps que cI est exprimé. Dans le cas du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], l’expression de cI n’est plus contrôlée par le promoteur pRM, mais induite par un [[Team:SupBiotech-Paris/BiobricksFr#drapeau|promoteur répressible LacP/O]] fusionné au gène cI. Si celui-ci n’est pas répressé, alors, comme dans le schéma naturel du bactériophage lambda, l’expression des gènes est inhibée.<br><br />
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En revanche, lorsque le signal de délivrance du vecteur cellulaire, la doxycycline, est injecté, alors le [[Team:SupBiotech-Paris/BiobricksFr#drapeau|promoteur répressible LacP/O]] est réprimé par le répresseur LacI, synthétisé par le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br />
cI inhibé, les gènes peuvent alors être transcrits, et le vecteur cellulaire synthétisé. <br><br />
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Système sans doxycycline:<br><br />
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Système avec doxycycline: <br><br />
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Lors de sa libération le vecteur cellulaire lyse le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]]. Ceci est un avantage, puisqu’il permet de détruire l’agent potentiellement pathogène du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]]. <br><br />
Une fois libéré dans son tissu cible, le vecteur cellulaire cible les cellules d’intérêts pour y délivrer le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Concept2Fr#drapeau|Haut de page]]</span><br />
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== Le système de ciblage cellulaire [1,2,9]==<br />
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Le vecteur cellulaire a pour but de cibler les cellules d’intérêt. Pour cela, l’ajout de protéine de ciblage cellulaire, au sein du génome du bactériophage lambda, est nécessaire.<br />
La délivrance du [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] se fait en deux étapes : le ciblage de la cellule d’intérêt et la libération au sein du cytoplasme.<br />
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=== Le ciblage de la cellule d’intérêt ===<br />
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Le bactériophage lambda interagit naturellement avec sa cellule cible, Escherichia Coli, via sa protéine de fibre de queue '''« J »'''. La protéine J étant l’unique protéine du bactériophage lambda intervenant dans la reconnaissance spécifique de la cellule hôte, elle est, par conséquent, la candidate idéale à une modification permettant de modifier le tropisme naturel du bactériophage.<br><br />
Pour se faire, la protéine J est greffée à une protéine possédant la caractéristique particulière de se lier de manière spécifique à l’un des récepteurs de la cellule cible. La protéine J interagissant par le coté Cter, c’est au niveau de ce dernier qu’est placé la protéine dite de fusion.<br><br />
Le gène de la protéine J est donc fusionné au niveau de l’extrémité 5’ avec le gène de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau| protéine de ciblage]]. La protéine qui en résulte, transcrite naturellement pendant la synthèse du vecteur cellulaire, est exprimée avec la même fréquence que la protéine native. De ce fait, lors de l’autoassemblage, le vecteur cellulaire possède la [[Team:SupBiotech-Paris/BiobricksFr#drapeau| protéine de ciblage]] sur sa queue.<br />
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En fonction du type cellulaire cible, le ligand peut changer, mais l’emplacement reste le même. Le [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] est par conséquent modulable à de nombreuses pathologies.<br />
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=== L’internalisation du vecteur cellulaire et la libération du plasmide thérapeutique ===<br />
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Le ciblage du vecteur cellulaire est assuré par la protéine fixée sur la protéine J, en revanche pour l’internalisation et la sortie de l’endosome, la fixation à un ligand membranaire ne suffit pas.<br />
C’est pourquoi, le vecteur cellulaire possède une seconde protéine recombinée. Elle se trouve, cette fois-ci, sur une autre protéine, non de queue mais de tête, la '''protéine D'''.<br />
[[Image :ProteinD.png|left|frameless|150px]]<br />
Le bactériophage lambda sauvage possède à sa capside, la protéine D qui est chargée de stabiliser l’ensemble de la capside, comme vu précédemment. Etant exposée sur toute la surface externe de la capside, elle est une candidate idéale pour la fusion d’une protéine d’internalisation cellulaire. <br />
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La protéine D, lors de sa fixation à la capside, est organisée en trimère. Chaque monomère est distant de 5,1nm dans un trimère. La structure cristallographique de la protéine D sous sa forme trimérique montre que les extrémités, Nter et Cter, sont exposées à la surface de la capside et sont très proches les unes des autres. <br />
Les deux extrémités Nter et Cter étant exposées à la surface de la capside mature, cela laisse le choix quant au placement de la protéine de fusion. Cependant des études menées en Nter et en Cter ont montré que la fusion en Cter apportait de meilleurs résultats.<br />
Cette protéine d’internalisation, nommée polypeptide de type III, est issue de la base du penton des adénovirus. En plus de permettre au vecteur cellulaire d’être internalisé dans la cellule d’intérêt, elle induit sa sortir de l’endosome.<br />
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[[Image :ADV5.png|center|frameless|600px]]<br />
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<div id="PB"><br />
La '''base du penton''' est une protéine pentamérique insérée stratégiquement au niveau des douze sommets de la capside adénovirale. Son monomère d’environ 60 kDa (571 aminoacides) est présent en 60 copies dans les virus. En plus de son rôle structural dans la capside, la base du penton possède une propriété fondamentale : elle contient un motif RGD (arginine - acide aspartique - glycine) conservé, responsable de l’interaction du virus avec les intégrines αvβ3 et αvβ5. Ce motif est situé à la surface de la base du penton et se projette vers l’extérieur de la capside.<br />
L'exposition de 5 motifs RGD portés par la base de penton (molécule pentamérique) permet l'association simultanée de plusieurs molécules d'intégrines, initiant l'entrée de l’adénovirus par endocytose, principalement au sein de vésicules à clathrines.<br />
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Le phénomène d’internalisation nécessitant le regroupement des intégrines via les motifs RGD, soit plusieurs monomères de polypeptides de type III, peut soulever un problème. En effet, la protéine D n’étant fusionnée qu’à un seul monomère de polypeptide de type III, l’endocytose peut ne pas avoir lieu.<br />
Cependant, d’après une étude cristallographique, la distance entre deux motifs RGD sur une base du penton est de 5,7nm. La distance entre deux protéines D étant similaire (5,1nm), le regroupement des intégrines peut avoir lieu et induire l’internalisation et la sortie de l’endosome.<br />
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Le bactériophage étant naturellement infectieux pour les organismes procaryotes, l’internalisation cellulaire et la sortie de l’endosome sont des facteurs limitant pour son utilisation au sein de modèles eucaryotes. Des études ont prouvé que la base du penton améliore de manière très significative l’internalisation cellulaire et la sortie de l’endosome.<br />
Sans base du penton, un bactériophage perd la quasi totalité de son efficacité de transfection. C’est un élément important pour le maintien d’une bonne efficacité de transfection du vecteur cellulaire.<br />
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L’ajout de la protéine d’internalisation, le polypeptide de type III, permet au vecteur cellulaire l’internalisation et la sortie de l’endosome des cellules eucaryotes. Une fois sortie de l’endosome, le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] est libéré au sein du cytoplasme de la cellule cible et peut ainsi agir.<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Concept2Fr#drapeau|Haut de page]]</span><br />
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== En résumé… ==<br />
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…, le [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] possède un vecteur cellulaire ayant les caractéristiques suivantes :<br />
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- '''Non pathogène''' donc pas toxique, <br><br />
- '''Ciblage cellulaire''', donc 2nde spécificité <br><br />
- '''Passage des membranes''' des cellules eucaryotes, <br><br />
-'''Encapsidation et délivrance de [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide spécifique]]'''. <br><br />
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Ces caractéristiques, spécifiques au [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]], apportent une solution aux problèmes récurrents, de [[Team:SupBiotech-Paris/Introduction1Fr#Spe|spécificité]] et de [[Team:SupBiotech-Paris/Introduction1Fr#PM|passage des membranes]] et de délivrance d’agent thérapeutique, rencontrés par les [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|vecteurs]].<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Concept2Team:SupBiotech-Paris/Concept22009-10-21T11:37:04Z<p>Aurel: /* The internalization of the cell vector and the release of the therapeutic plasmid */</p>
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= Cell vector =<br />
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The cell vector has to reach the target cell and delivers [[Team:SupBiotech-Paris/Concept3#drapeau|the therapeutic gene insert]].<br><br />
To be an efficient targeting cell vector, he has to get several important characteristics. He has to target specifically the interest cell type, the one to cure. As a phage vector, he has to be able to cross the eukaryotic cell membrane and deliver his content in an efficient way. <br><br />
You are going to discover, across this chapter, what kind of characteristics have been decisive in order to choose the '''best bacteriophage'''.<br><br />
Then, you are going to discover, in details, its properties according to the [[Team:SupBiotech-Paris/Concept#DVS|DVS]] characteristics: '''tissue deliverance''', '''[[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] encapsidation''', and '''cell targeting'''.<br><br />
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==The cell vector: The lambda phage [1,2]==<br />
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We have studied several kind of phages, in order to select the cell vector the most controllable, and adaptable. So, we have identified all the different kind of phages, and in one kind of phage, the species the most described. Several candidates have retained our attention, but it is the lambda phage which seems to be the most adapted.<br><br />
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The lambda phage is a prokaryotic virus, which infects the bacteria ''Escherichia coli''. This is a tempered phage, because it can alternate lytic and lysogenic cycle under some conditions. During the lytic cycle, the circular DNA is replicated in great quantity in the cytoplasm; a lot of viral progeny are formed, driving to the cell death and the virions liberation. During the lysogenic cycle, the phage genome is integrated in the bacterial chromosome, and is transmitted to the line of descent. The replication is achieved at the rythm of the bacterial cell division.<br><br />
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Naturally infectious for the bacteria ''Escherichia coli'', the lambda phage, as every phage, is not infectious for man. This characteristic comes from the fact that the genome is governed by prokaryotic promoters but also because it has a very low capacity to transfect eukaryotic cells.<br><br />
Finally, one of the most interesting characteristics of the lambda phage is the fact that he has a linear double stranded DNA, with cohesive ends: 12 nucleotides complementary “sticky” ends, called “Cos” sequence. These cohesive ends have a '''genomic encapsidation''' function.<br><br />
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==The encapsidation of the therapeutic plasmid [2,3,4,5,6,7]==<br />
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The encapsidation of the viral genome is particular for the lambda phage. It is managed by the [[Team:SupBiotech-Paris/Concept3#cos|Cos sequence]]. This characteristic allows the specific encapsidation of the sequence of interest.<br><br />
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[[Image:Encapsidation.png|center|650px|frameless]]<br />
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We can observe in this picture that the initiation and the terminaison of the encapsidation is mediated by the [[Team:SupBiotech-Paris/Concept3#cos|Cos sequence]]. This is the fragment between two [[Team:SupBiotech-Paris/Concept3#cos|Cos sequences]] which is encapsidated. This particularity is used in molecular biology since several years in order to create DNA banks and molecular tools named Cosmides.<br><br />
According to this fact, we decided to encapsidate, inside the cell vector, only the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]]. So, the Cos sequence of the lambda genome is deleted, and are placed in [[Team:SupBiotech-Paris/Concept3#drapeau|the therapeutic plasmid]].<br><br />
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Non-coding sequence Cos: Position 1-132 et 48462-48502 – length: 174 bp.<br><br />
1.....TATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTAC<br><br />
41....GGGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATT<br><br />
81....TTCCGGTTTAAGGCGTTTCCGTTCTTCTTCGTCATAACTT<br><br />
121...AATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAAC<br><br />
161...GACAGGTGCTGAA<br><br />
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[[Image :Schémades2plasmides|center|frameless]]<br />
Schéma des deux plasmides : génome de lambda recombiné + plasmide d’intérêt<br />
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The cell vector encapsulates the therapeutic plasmid inside its capsid because of the [[Team:SupBiotech-Paris/Concept3#cos|Cos sequence]]. Thus, it can deliver the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic agent]] in the cells, which allows a supplementary precaution, in case of the interest vector is not inside the target organ.<br><br />
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<div id="C1"></div><br />
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==The cell vector deliverance in the target organ [1,2,8]==<br />
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The cell vector has two actions phase, a '''latency''' phase and an '''expression and release''' phase. During the latency phase, called '''lysogenic cycle''', the genome stays “inactive” and no cell vector is produced inside the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]]. Whereas, during the expression and release phase, called '''lytic cycle''', the genome is transcribed and replicated, in order to produce the cell vectors.<br><br />
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However, the tissue vector is not the natural host of the cell vector. So, if we want to induce a latency phase, we have to cause it. The deliverance system introduced into the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]] is designed to induce this latency phase.<br><br />
The cell vector plasmid of the deliverance control induces a lysogenic cycle of this one.<br><br />
The lysogenic cycle is a phenomenon naturally present in the lambda phage, the cell vector. During this cycle, the viral genome is inserted into the bacterial genome as a prophage, where every transcription is repressed. Only the cI gene, responsible of the lysogenic cycle maintain, is expressed.<br><br />
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The cI fixation on the operators under the control of pR and pL promoters, inhibited the genes expression and allows the lysogeny maintain. cI inhibited also its own promoter, pRM, allowing an autoregulation of its transcription.<br><br />
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The phage is maintained under the lysogenic form as long as cI is expressed. In the case of the [[Team:SupBiotech-Paris/Concept#DVS|DVS]], the expression of cI is not controlled by the pRM promoter, but induced by the [[Team:SupBiotech-Paris/Biobricks#drapeau|repressible promoter LacP/O]] fused to the cI gene. If this one is not repressed, the gene expression is inhibited, as the lambda phage natural pathway.<br><br />
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But, as soon as the deliverance signal of the cell vector, the doxycyclin, is injected, the [[Team:SupBiotech-Paris/Biobricks#drapeau|LacI repressor]], synthesized by the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]], represses the [[Team:SupBiotech-Paris/Biobricks#drapeau|repressible promoter LacP/O]]. cI inhibited, the genes can be transcribed, and the cell vector synthesized.<br><br />
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System without doxycyclin:<br> <br />
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As soon as the cell vector is synthesized and released, the cell vectors lyse the [[Team:SupBiotech-Paris/Concept1#drapeau|tissue vector]]. This is an advantage, because it allows the destruction of the potentially pathogen agent of the [[Team:SupBiotech-Paris/Concept#DVS|DVS]].<br><br />
Moreover, no sooner, the [[Team:SupBiotech-Paris/Concept2#drapeau|cell vector]] is released in the target tissue, the cell vector targets the interest cells in order to deliver the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
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==The system of cell targeting [1,2,9]==<br />
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The cell vector is used in order to target the interest cells. The adding of cell targeting proteins, inside the lambda phage genome, is necessary.<br><br />
The deliverance of the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] is achieved in two steps: the cell targeting and the cytoplasmic release.<br><br />
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=== The interest cell targeting ===<br />
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The lambda phage naturally interacts with its target cell, ''Escherichia coli'', via its tail fiber '''protein J'''. Only the protein J mediates the specific recognition of its bacterial host. It is, consequently, the ideal candidate to a modification which allows a change in the natural tropism of the phage.<br><br />
In order to do this, the protein J is fused to a protein, which is able to bind specifically to a targeted cell receptor. The protein J interacts with the ''Escherichia coli'' membrane receptor by the Cter end that is why, we decided to bind a fusion protein to this side.<br><br />
The protein J gene is fused at the 5’ end to the targeting protein gene. The protein which results of this construction, naturally transcribed during the cell vector synthesis, is expressed with the same frequency as the native protein. So, during the self-assembly, the cell vector has a targeting protein on his tail.<br><br />
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According to the targeted cell type, the ligand can change, but the location, next the protein J, stays the same. The [[Team:SupBiotech-Paris/Concept#DVS|DVS]] is consequently adaptable to several pathologies.<br><br />
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=== The internalization of the cell vector and the release of the therapeutic plasmid ===<br />
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The protein fused to the J protein mediates the targeting of the cell vector, but, for the internalization and the endosomal escape, the fixation of a membrane ligand is not sufficient. That is why, the cell vector have a second recombinant protein. This one is fused to another protein, not on the tail but on the head of the phage, the '''protein D'''.<br />
[[Image :ProteinD.png|left|frameless|150px]]<br />
The lambda phage wild type has on the capsid, the protein D, which is in charge of the stabilization of the entire capsid, as we saw previously. The protein D is exposed on the entire outer surface of the capsid, that is why, it is an ideal candidate to be fused to a cellular internalization protein.<br><br />
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The D protein is organized as a trimer. Each monomer is distant of 5,1nm in a trimer. The crystal structure of the protein D, as a trimeric form, shows that the Nter and Cter ends, are exposed at the capsid surface, and are very closed from each other. The Nter and Cter ends are exposed at the mature capsid surface, which allows the choice for the location of the fusion protein (Nter or Cter). However, several studies have showed that the fusion to the Cter end, allows better results.<br><br />
The internalization protein, called polypeptide III, is issued of the adenovirus penton base. This one allows to the cell vector to be internalized in the interest cell, and to induce the endosomal escape.<br><br />
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[[Image :ADV5.png|center|frameless|600px]]<br />
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<div id="PB"><br />
The '''penton base''' is a pentameric protein strategically inserted in the twelve peaks of the adenoviral capsid. Its monomer, which the length is around 60 kDa (571 aminoacides), is present in 60 copies in the viruses. More than its structural function in the capsid, the penton base has a fundamental property: it contains an RGD motif (arginin - aspartic acid - glycin) conserved, responsible of the interaction of the virus with the integrins αvβ3 and αvβ5. This motif is located at the penton base surface and is exposed to the outer of the capsid.<br><br />
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The exposition of 5 RGD motifs carries by the penton base, allows the simultaneous association of several integrin molecules, which initiate the incorporation of the adenovirus by endocytosis, principally mediated by clathrin vesicles.<br><br />
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The integrins gathering via the RGD motif is necessary for the internalization phenomenon. The gathering needs to several polypeptides III monomers. This can reveal a problem, indeed, only one monomer of polypeptide III is fused to one protein D, so the endocytosis could not happen.<br><br />
However, since a crystallography study, the distance between two RGD motifs on one penton base is equal to 5,7nm. The distance between two proteins D is similar (5,1nm), so the integrins gathering can be achieved and induces the internalization and the endosomal escape.<br><br />
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Because of the fact that the lambda phage is naturally infectious for the prokaryotic organisms, the cellular internalization and the endosomal escape are serious limiting factors for its use to transfect eukaryotic cells. But, some studies have shown that the penton base improves in a significant way the cellular internalization and the endosomal escape.<br><br />
Without any penton base, a phage looses almost the totality of its transfection efficiency. It is an important element for the maintain of a good transfection efficienty of the cell vector.<br><br />
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The adding of the internalization protein, the polypeptide III, allows to the cell vector, the internalization and the endosomal escape of the eukaryotic cells. Once outside of the endosome, the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] is released in the cytoplasm of the targeted cell and can act.<br><br />
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==To summarize…==<br />
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The [[Team:SupBiotech-Paris/Concept#DVS|DVS]] has a cell vector , with the following characteristics: <br><br />
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- '''Non pathogenic''' so not toxic,<br><br />
- '''Cell targeting''', so the 2nd specificity,<br><br />
- '''Passage of the eukaryotic cells membrane''',<br><br />
- '''Encapsidation and delivery of a [[Team:SupBiotech-Paris/Concept3#drapeau|specific plasmid]]'''.<br><br />
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This characteristics, specific to the [[Team:SupBiotech-Paris/Concept#DVS|DVS]], bring a solution to the redundant problems, of [[Team:SupBiotech-Paris/Concept#Spe|specificity]], [[Team:SupBiotech-Paris/Concept#PM|membrane passage]], and therapeutic agent delivery, encounter by the [[Team:SupBiotech-Paris/Concept#drapeau|vectors]]. <br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T11:34:49Z<p>Aurel: </p>
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= Cell targeting =<br />
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== Context ==<br />
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After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
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==Objective ==<br />
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Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a [[Team:SupBiotech-Paris/Concept2#PB| penton base]] from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
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== Experimental approach ==<br />
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In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 [[Team:SupBiotech-Paris/Concept2#PB| penton base]] extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
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Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]], amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 5 and 6). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the [[Team:SupBiotech-Paris/Concept2#PB| penton base]] is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
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<a href="https://2009.igem.org/Team:SupBiotech-Paris/Treatement_modeling#drapeau" target="_self"><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T11:29:51Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#PB| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a penton base from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 penton base extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the penton base with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the penton base, amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the penton base and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the penton base forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 5 and 6). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the penton base used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus penton base is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the penton base complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the penton base alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the penton base (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the penton base is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
<br />
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<br />
<br />
<br />
<br />
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<a href="https://2009.igem.org/Team:SupBiotech-Paris/Treatement_modeling#drapeau" target="_self"><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T11:29:05Z<p>Aurel: </p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The [[Team:SupBiotech-Paris/Concept2#7| penton base]] originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a penton base from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 penton base extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the penton base with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the penton base, amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the penton base and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the penton base forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 5 and 6). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the penton base used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus penton base is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the penton base complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the penton base alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the penton base (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the penton base is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
<br />
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<br />
<br />
<br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T11:25:38Z<p>Aurel: /* Results */</p>
<hr />
<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The penton base originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a penton base from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 penton base extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the penton base with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the penton base, amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the penton base and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the penton base forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 5 and 6). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the penton base used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus penton base is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the penton base complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the penton base alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the penton base (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the penton base is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
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[[image: gènes mutés.jpeg]]<br />
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These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
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Here is the allocation planning of electroporations: <br><br />
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[[image:planning.jpeg]] <br />
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Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
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Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
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However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
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Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
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A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<br />
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<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
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In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
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<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
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Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
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Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
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<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
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<br />
<br />
<br />
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<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
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After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
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<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
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<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
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<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
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== Conclusion [3,4,5,6,7,8,9] == <br />
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Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
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The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T11:16:19Z<p>Aurel: /* Results */</p>
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<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The penton base originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a penton base from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 penton base extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the penton base with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the penton base, amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the penton base and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the penton base forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
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Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 5 and 6). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
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Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the penton base used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus penton base is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the penton base complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the penton base alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the penton base (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the penton base is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
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These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
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<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T11:13:24Z<p>Aurel: </p>
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<div>{{Template:Supbiotechcss.css}}<br />
{{Template:SupbiotechparisEn}}<br />
<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, because of its high capacity of cloning and a capsid structure adapted to a concentrated presence of exogenous proteins, is a good candidate to design an eukaryotic [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]]. The penton base originally from the adenovirus capsid appears as a promising candidate for Lambda phage targeting. Indeed, it is endowed of several functions like the cell receptors link, the viral particles internalisation and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
<br />
Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a penton base from the adenovirus 5 fused by its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Cell targeting#drapeau|Back to top]]</span><br />
<br />
<br />
== Experimental approach ==<br />
<br />
In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage. The protein D extraction from Lambda phage genome has been lead by Polymerase Chain Reaction (PCR) with several couple of primers. The same strategy has been applied for the adenovirus 5 penton base extraction from which has been extracted a plasmid coding for the virus offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
After the fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistance against an antibiotic to confirm the transfection of the recombined phage into bacteria and a reporter gene, like GFP, with eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
<br />
Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]], a Lambda phage type, confection is possible by fusion of the penton base with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the penton base, amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Results ==<br />
<br />
=== Design of the fusion protein ===<br />
<br />
For the fusion protein design, we decided to extract separately the penton base and the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the Lambda phage thanks to primers containing a BalI restriction site on the [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the penton base forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
<br />
<br />
First and second pair’s genes extraction: <br><br />
<br />
<br />
D protein of the Lambda phage: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Adenovirus 5 penton base : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base (sample number 9) and 385bp for the D protein (samples 5 and 6). However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR of D protein BioBrick (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
=== Transfection of eukaryotic cells by the Lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
<br />
A cytofluorimetric study has been done to analyze the transfection rate of recombined Lambda phages. Figure X shows cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyze of the GFP fluorescence on non recombined Lambda phages (Lambda), recombined Lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined Lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
<br />
<br />
Firstly, we observe that the recombined phage shows a tag difference independently of the fragment of the penton base used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Discussion ==<br />
<br />
Even if the tissue vector has not been finished, scientific literature shows that a recombinant phage creation with a protein coding the adenovirus penton base is possible. It demonstrates as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the penton base complete fragment (figure 2). In the case of our application it is possible to use a recombined Lambda phage to insert our therapeutic gene. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
== Conclusion ==<br />
<br />
To conclude the RGD fragment of the penton base alone has a higher efficiency of interaction with integrines of eukaryotic cells. However for our project, it was more judicious to use the complete sequence of the penton base (fragment 1-571) because the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give a very fast and target injection of bacteriophages. The use of a highly efficient transfection system is not advised because phages do not have the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the penton base is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
<br />
= Application =<br />
== Context ==<br />
<br />
In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
<br />
The [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
<br />
The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
<br />
<br />
[[image: gènes mutés.jpeg]]<br />
<br />
<br />
These different genes play a predominant role in the application of the apoptotic process and are the most susceptible to be mutated in the lung cancer case. They compose the [[Team:SupBiotech-Paris/Concept3#drapeau|therapeutic plasmid]].<br><br />
<br />
== The objective ==<br />
<br />
The objective of this study is to check if a wild type version of a tumor suppressor gene inside the tumor cell, for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
<br />
== Experimental approach ==<br />
<br />
<br />
=== Cancer cell line and reported gene ===<br />
<br />
We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
<br />
<br />
<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
<br />
<br />
=== TP53 gene incorporation ===<br />
<br />
Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
<br />
<br />
<br />
<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
</ul><br />
<br />
<br />
<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
<br />
<br />
=== Apoptosis detection ===<br />
<br />
Detection of apoptotic cells is done by the annexin V assay: <br><br />
<br />
In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. This is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analyzed by flow cytometry. <br><br />
<br />
<br />
<br />
<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
<br />
<br />
Work as much as possible in the dark (fluorophores are photolabile) <br><br />
<br />
<br />
<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The running of the study ==<br />
<br />
The time of the plasmid expression in DU-145 cell line was not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
<br />
Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
<br />
<br />
Here is the allocation planning of electroporations: <br><br />
<br />
<br />
[[image:planning.jpeg]] <br />
<br />
<br />
Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyze. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
<br />
<br />
Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
<br />
<br />
However, several studies showed that to bring p53 wild type into tumor mutated cells launch the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who was working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type were not transfected by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after the infection of a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agar gel of their genome has been realized.<br><br />
<br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptotic process. <br><br />
<br />
A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
<br />
<br />
<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induces the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
<br />
<br />
<br />
Similar results were reported in the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
<br />
<br />
<dt> Morphologic analyze of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
<br />
<br />
<br />
Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
<br />
<br />
<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
<br />
<br />
<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
<br />
<br />
<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
<br />
<br />
<dd><u><i>This study show one more time that to bring a p53wild-type version into a mutated p53 cell population induces cell death by apoptosis.</i></u><br><br />
<br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Even if we could not give the proof by our own experiments, many studies show that to bring a wild-type version of a tumor suppressor gene into a mutated tumor cell for this gene permits to launch the apoptosis. <i>''In vivo''</i> studies on Human in the framework of the prostate, ovary and lung cancers have already been hold and present convincing results. <br><br />
<br />
The implementation of this study has been originally done to determine if the [[Team:SupBiotech-Paris/Introduction1#drapeau|DVS]] application in the fight against non small cell lung cancer is feasible or not. Because we have not been able to conclude, the implementation of the study has been done by analyzing several publications. According to these publications, the application is first confirmed in the framework of the chosen pathology but it can also be reached to others cancers like hepatocellular carcinoma, on which the fact to bring a gene suppressor of tumor launch the apoptosis process. The only limitation is set by the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] tropism.< br><br />
<br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T10:42:45Z<p>Aurel: /* Conclusions */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes. Cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée des bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
<br />
C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
<br />
[[image: gènes mutés.jpeg|center]]<br />
<br />
Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== L’objectif ==<br />
<br />
L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
<br />
== Démarche expérimentale ==<br />
<br />
<br />
=== Lignée cancéreuse et gène apporté ===<br />
<br />
Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Incorporation du gène TP53 ===<br />
<br />
L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
<br />
<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T10:31:57Z<p>Aurel: /* Design de la protéine de fusion */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 et la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
<br />
C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
<br />
[[image: gènes mutés.jpeg|center]]<br />
<br />
Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== L’objectif ==<br />
<br />
L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
<br />
== Démarche expérimentale ==<br />
<br />
<br />
=== Lignée cancéreuse et gène apporté ===<br />
<br />
Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Incorporation du gène TP53 ===<br />
<br />
L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
<br />
<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T10:16:14Z<p>Aurel: /* Contexte */</p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
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Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
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=== Design de la protéine de fusion ===<br />
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Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
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Base de penton de l’adénovirus 5 : <br><br />
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Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
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Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
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Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
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Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Discussion ==<br />
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Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusions ==<br />
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Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
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= Le Plasmide antitumoral =<br />
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== Contexte ==<br />
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Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
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<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
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La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
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En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats [1,2] ==<br />
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Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
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<br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
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<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T09:45:53Z<p>Aurel: /* Conclusion [3,4,5,6,7,8,9] */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
<br />
C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
<br />
[[image: gènes mutés.jpeg|center]]<br />
<br />
Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== L’objectif ==<br />
<br />
L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
<br />
== Démarche expérimentale ==<br />
<br />
<br />
=== Lignée cancéreuse et gène apporté ===<br />
<br />
Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Incorporation du gène TP53 ===<br />
<br />
L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
<br />
<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
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<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
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<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
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<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
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La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T09:43:38Z<p>Aurel: /* Conclusion [3,4,5,6,7,8,9] */</p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
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= Le Ciblage cellulaire =<br />
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== Contexte ==<br />
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Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
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=== Design de la protéine de fusion ===<br />
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Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
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Base de penton de l’adénovirus 5 : <br><br />
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Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
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Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
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Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
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Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Discussion ==<br />
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Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusions ==<br />
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Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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= Le Plasmide antitumoral =<br />
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== Contexte ==<br />
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Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
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<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
<br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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<a href="https://2009.igem.org/Team:SupBiotech-Paris/Modeling_du_traitement#drapeau" target="_self"><br />
<img title="On passe à la page suivante !" style="width: 100px;" src="https://static.igem.org/mediawiki/2009/e/e9/Suivant.png";><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T09:43:15Z<p>Aurel: /* Conclusion [3,4,5,6,7,8,9] */</p>
<hr />
<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
<br />
== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
<br />
<br />
Première et deuxième paires pour l’extraction des gènes : <br><br />
<br />
<br />
Protéine D du phage Lambda: <br><br />
<br />
Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
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La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
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En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats [1,2] ==<br />
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Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
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<br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
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<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
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<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
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<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusion [3,4,5,6,7,8,9] == <br />
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Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
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La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T09:42:21Z<p>Aurel: /* Conclusion [3,4,5,6,7,8,9] */</p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
<br />
= Le Ciblage cellulaire =<br />
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== Contexte ==<br />
<br />
Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
<br />
==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
<br />
Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
<br />
=== Design de la protéine de fusion ===<br />
<br />
Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
<br />
Base de penton de l’adénovirus 5 : <br><br />
<br />
Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
<br />
<br />
Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
<br />
<br />
Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
<br />
<br />
Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
<br />
<br />
Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
<br />
Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
<br />
[[image:M2109.png|center]]<br />
<br />
<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
<br />
Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
<br />
[[image:VT1.png|center]]<br />
<br />
[[image:VT2.png|center]]<br />
<br />
<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
<br />
<br />
Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Discussion ==<br />
<br />
Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusions ==<br />
<br />
Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
= Le Plasmide antitumoral =<br />
<br />
== Contexte ==<br />
<br />
Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
<br />
L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
<br />
C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
<br />
[[image: gènes mutés.jpeg|center]]<br />
<br />
Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== L’objectif ==<br />
<br />
L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
<br />
== Démarche expérimentale ==<br />
<br />
<br />
=== Lignée cancéreuse et gène apporté ===<br />
<br />
Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
<br />
<br />
<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Incorporation du gène TP53 ===<br />
<br />
L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
<br />
<br />
<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
<br />
<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
=== Détection de l’apoptose ===<br />
<br />
La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
<br />
En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
<br />
<br />
<br />
<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
<br />
<br />
Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
<br />
<br />
<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Déroulement de l’étude ==<br />
<br />
Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
<br />
N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
<br />
<br />
Voici le planning de répartition des électroporations: <br><br />
<br />
[[image:planning.jpeg|center]] <br />
<br />
<br />
Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
<br />
La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
<br />
En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Résultats [1,2] ==<br />
<br />
Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
<br />
<br />
N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
<br />
<br />
Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
<br />
<br />
[[image:figure 2bis.jpeg|float|left]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
<br />
Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
<br />
<br />
<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
<br />
<br />
<br />
Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
<br />
<br />
<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
<br />
<br />
<br />
Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
<br />
<br />
<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
<br />
<br />
<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
<br />
<br />
<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
<br />
<br />
<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
<br />
<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
<br />
La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
<br />
<br />
<br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T09:41:51Z<p>Aurel: /* Résultats [1,2] */</p>
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= Le Ciblage cellulaire =<br />
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== Contexte ==<br />
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Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
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==Objectif ==<br />
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Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Démarche expérimentale ==<br />
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Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
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Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
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=== Design de la protéine de fusion ===<br />
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Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
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Base de penton de l’adénovirus 5 : <br><br />
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Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
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Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
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Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
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Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Discussion ==<br />
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Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusions ==<br />
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Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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= Le Plasmide antitumoral =<br />
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== Contexte ==<br />
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Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
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<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
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La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
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En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats [1,2] ==<br />
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Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
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<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
<br />
<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
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<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
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<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
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<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
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La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Ciblage_CellulaireTeam:SupBiotech-Paris/Ciblage Cellulaire2009-10-21T09:40:39Z<p>Aurel: /* Conclusion [3,4,5,6,7,8,9] */</p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisFr}}<br />
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= Le Ciblage cellulaire =<br />
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== Contexte ==<br />
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Après l’action du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]], viens le [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]], celui-ci est un bactériophage modifié qui a la faculté d’infecter les cellules eucaryotes. Le bactériophage lambda, du fait de sa grande capacité de clonage et une structure de capside adaptée à une présence concentrée de protéines exogènes, est un très bon candidat pour le design d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] eucaryote. La base de penton issue de la capside de l’adénovirus apparait comme un candidat prometteur pour le ciblage du phage lambda. En effet, elle est dotée de plusieurs fonctions telles que la liaison aux récepteurs cellulaires, l’internalisation des particules virales et la libération de la capside par l’endosome.<br><br />
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==Objectif ==<br />
<br />
Nos objectifs sont de designer un [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] de type bactériophage Lambda recombiné avec une base de penton issue de l’adénovirus 5 fusionnée à sa [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]]. Le [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] doit être capable d’intégrer la cellule, sortir de l’endosome, transporter son ADN vers le noyau de la cellule et finalement transcrire ce(s) [[Team:SupBiotech-Paris/Concept3Fr#drapeau|gène(s) thérapeutique(s)]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Démarche expérimentale ==<br />
<br />
Dans le cadre du design des gènes du bactériophage recombinant nous avons décidé de fusionner la base de penton de l’adénovirus 5 avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du phage lambda. L’extraction de la protéine D à partir du génome du bactériophage Lambda a été menée par réaction de polymérisation en chaine (PCR) avec plusieurs paires de primers. La même stratégie a été prise pour l’extraction de la base de penton de l’adénovirus 5 qui a été extraite d’un plasmide codant pour le virus gracieusement donné par le Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
<br />
Après la formation de la protéine de fusion, celle-ci est introduite dans un plasmide BioBrick. Le plasmide contient une résistance contre un antibiotique pour la confirmation de la transfection du phage recombiné dans la bactérie. Ainsi qu’un gène rapporteur tel que la GFP avec un promoteur eucaryote, le CMV du <i>Simian virus</i> 40 (SV40), pour confirmer la transfection dans les cellules eucaryotes. Cette stratégie nous permet alors de prouver que le bactériophage est capable d’infecter les cellules eucaryotes. <br><br />
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Malheureusement nous n’avons pas été capable de construire la protéine de fusion dans le temps requis. Cependant la littérature scientifique démontre que la confection d’un [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]] type bactériophage lambda est possible par fusion de la base de penton de l’adénovirus avec la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] (Stefania Piersanti et al. 2004). La séquence centrale de la penton base, acides aminés 1 à 571, fusionnée avec le bactériophage offre une transfection dans les cellules eucaryotes, tous comme l’utilisation du fragment RGD responsable de l’entrée du virus et la sortie de l’endosome, fragment 286 à 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats ==<br />
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=== Design de la protéine de fusion ===<br />
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Pour le design de la protéine de fusion, nous avons décidé d’extraire séparément la base de penton de l’adénovirus 5 à la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] du bactériophage lambda grâce à des primers qui contiennent un site de restriction BalI sur le primer reverse de la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] et le primer forward de la base de penton. De plus la protéine de fusion finale contient les fragments spécifiques aux BioBricks à ces deux extrémités. <br><br />
Pour l’extraction des 2 gènes nous avons utilisé les primers suivants : <br><br />
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Première et deuxième paires pour l’extraction des gènes : <br><br />
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Protéine D du phage Lambda: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
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Base de penton de l’adénovirus 5 : <br><br />
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Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
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Troisième paire pour l’extention du site de restriction BalI et du préfixe BioBrick pour la [[Team:SupBiotech-Paris/BiobricksFr#drapeau|protéine D]] seulement (déjà effectué pour la base de penton). <br><br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Quatrième paire pour l’amplification de la protéine de fusion après ligation des deux fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
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Les résultats de PCR sont présentés dans la figure X. Nous observons qu’il y a bien amplification de fragments qui correspondent aux tailles de la base de penton = 1715bp pour l’échantillon 9 et la de protéine D = 385bp pour l’échantillon 5 et 6. Il y a cependant beaucoup de phénomènes de mismatch pendant les cycles d’amplification. Cela pourrait avoir un effet négatif sur le résultat d’amplification final. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR des BioBricks de la protéine D (1, 2, 3) et de la base de penton (4, 5, 6), de la protéine D (5 et 6) et de la base de penton (7, 8, 9) avec les sites BalI </i><br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Transfection des cellules eucaryotes par le phage lambda recombiné avec la base de penton fusionnée à la protéine D (Stefania Piersanti et al., 2004) ===<br />
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Une étude au par cytofluorimétrie a été faite afin d’analyser le taux de transfection des bactériophages lambda recombinés. La figure X montre les résultats de cytofluorimétrie de l’analyse de cellules COS-1 après avoir été exposées à une concentration de 10^6 PFU/cellules de phages recombinants, Pb (1-571) ou Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyse de la fluorescence de la GFP sur des phages lambda non recombinés (Lambda), des phages lambda recombinés avec le fragment 286-393 de la base de penton (LambdaPb286-393), des phages lambda recombinés avec la base de penton complète (1-571), des adénovirus marqués à la GFP (Ad10 et Ad100)</i><br><br />
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Premièrement, nous observons que le phage recombiné montre bien une différence de marquage quelque soit le fragment de base de penton utilisé comparé au bactériophage non transformé. Secondement, le phage recombiné avec le fragment RGD seul (286-393) à une fluorescence plus élevée que le phage avec un fragment complet et plus proche de celui des adénovirus (figure X). <br><br />
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== Discussion ==<br />
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Bien que le vecteur tissulaire n’ait pas été fini, la littérature scientifique montre que la création d’un phage recombiné avec une protéine codant la base de penton de l’adénovirus est possible. Il est aussi démontré que les fragments codant pour les séquences RGD seuls ont une plus forte capacité à infecter les cellules eucaryotes comparé au fragment complet de la base de penton (figure 2). Dans le cas de notre application il est alors possible d’utiliser un bactériophage lambda recombiné pour insérer notre gène thérapeutique. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Conclusions ==<br />
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Pour conclure le fragment RGD seul de la base de penton a la meilleure efficacité d’interaction avec les intégrines des cellules eucaryotes, cependant dans le cadre de notre projet il est plus judicieux d’utiliser la séquence complète de la base de penton (fragment 1-571) car avec l’utilisation du [[Team:SupBiotech-Paris/Concept2Fr#drapeau|vecteur cellulaire]] et du système d’induction par la doxycycline donne une injection très rapide et très ciblée de nos bactériophages. L’utilisation d’un système de transfection hautement efficace est déconseillé car les phages n’ont pas le temps de se disperser correctement et vont alors infecter plusieurs fois la même cellule. L’utilisation du fragment complet de la base de penton est suffisant pour que le phage infecte correctement les cellules eucaryotes et lui laisse le temps d’avoir une dispersion plus que correct. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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= Le Plasmide antitumoral =<br />
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== Contexte ==<br />
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Dans le cancer du poumon non à petites cellules, ou NSCLC, comme dans tous cancers, la perte de la capacité apoptotique des cellules tumorales est du à la perte fonctionnelle de divers suppresseurs de tumeur entrant dans la voie de signalisation de la cascade apoptotique.<br><br />
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L’application du [[Team:SupBiotech-Paris/Introduction1Fr#DVS|DVS]] dans la lutte anti-cancer repose sur le fait de réactiver cette cascade apoptotique en apportant au sein des cellules tumorales une version wild-type des gènes codant les suppresseurs de tumeur non-fonctionnels.<br><br />
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C’est le [http://www.sanger.ac.uk/genetics/CGP/cosmic/ projet COSMIC] de [http://www.sanger.ac.uk/ l’institut Sanger] qui nous a permis de déterminer quels gènes apporter au [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]] dans le cadre du cancer du poumon à non petites cellules. Ce projet répertorie en effet toutes les mutations détectées pour chaque type de cancers suivant leur fréquence d’apparition. Ainsi, d’après leurs données, la perte de la capacité apoptotique des cellules tumorales pour un cancer du poumon peut être du à la perte fonctionnelle des protéines issus des gènes suivant :<br><br />
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[[image: gènes mutés.jpeg|center]]<br />
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Ces différents gènes, jouant un rôle prépondérant dans la mise en place du processus apoptotique et étant les plus susceptibles d’avoir mutés dans le cadre d’un cancer du poumon, compose le [[Team:SupBiotech-Paris/Concept3Fr#drapeau|plasmide thérapeutique]].<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== L’objectif ==<br />
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L’objectif de cette étude est de vérifier si le fait d’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale pour qui sa version est mutée, induit ou pas le phénomène d’apoptose.<br><br />
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== Démarche expérimentale ==<br />
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=== Lignée cancéreuse et gène apporté ===<br />
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Nous avons sélectionné parmi les lignées cellulaires qui étaient à notre disposition, une lignée cancéreuse dont l’origine cancéreux était du à la mutation d’un gène suppresseur de tumeur. La version wild-type du gène TP53 étant en notre possession, c’est la lignée cancéreuse prostatique p53 muté DU-145 qui retint notre attention.<br><br />
Nous allons donc tester si le fait d’amener une version wild-type de la protéine p53 (p53wt) au sein de la lignée DU-145 permet le déclenchement du processus d’apoptose.<br><br />
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<i>Protocole de mise en culture : </i><br><br />
<ol><br />
<li>Sortir l’ampoule de l’azote liquide<br><br />
<li>Placer l’ampoule dans un bain-marie à 37°C pendant 5 minutes<br><br />
<li>Dans un falcon 50 ml, mettre 9 ml de MEM 10% + 1 ml d’ampoule<br><br />
<li>Centrifuger 5 min à 1200 rpm<br><br />
<li>Aspirer le surnageant sans toucher aux cellules culotées (élimination du DMSO) <br><br />
<li>Resuspendre le culot dans 1 ml de milieu<br><br />
<li>Déposer le tout dans une nouvelle flasque T25 contenant 5 ml de milieu<br><br />
<li>Incubation à 37°C<br><br />
<li>Ne pas oublier de changer le milieu le lendemain pour éliminer les traces de DMSO<br><br />
<li>Après une semaine, les cellules sont à confluence 100%<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Incorporation du gène TP53 ===<br />
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L’incorporation du plasmide contenant p53wt, pcDNA3 CMV+p53wt, au sein des cellules DU-145 s’est effectuée par électroporation. <br><br />
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<i>Matériel :</i> <br><br />
<ul><br />
<li>Cellules DU-145<br><br />
<li>Plasmide pcDNA3 CMV+p53wt<br><br />
<li>Milieu de culture électrocompétent<br><br />
<li>Trypsine<br><br />
<li>PBS<br><br />
<li>Bac à glace<br />
<li>Cuvette d’électrotransfert<br />
<li>Centrifugeuse<br />
<li>Incubateur<br />
<li>Electroporateur (cliniporateur)<br />
</ul><br />
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<i>Protocole: </i> <br><br />
<ol><br />
<li>Aspirer le milieu du T25 contant les DU-145<br><br />
<li>Rincer au PBS<br><br />
<li>Déposer 500 µl de trypsine et laisser agir 3 minutes à température ambiante<br><br />
<li>Ajouter 5 ml de MEM 10% pour neutraliser la trypsine<br><br />
<li>Suspendre les cellules<br><br />
<li>Récupérer le milieu contenant les DU-145 dans un tube et centrifuger à 1000rpm pendant 10 minutes<br><br />
<li> Aspirer le surnageant et resuspendre le culot dans Xµl (X= 90µl x Nombre de cuves) de milieu électrocompétent (environ 5x105 cellules par cuves) <br><br />
<li>Suspendre votre solution d’ADN dans du milieu électrocompétent (18x10-2g/L) <br><br />
<li>Ajouter 10µl de solution d’ADN par cuve<br><br />
<li>Ajouter 90µl de la suspension cellulaire<br><br />
<li>Mettre les cuves dans la glace<br><br />
<li>Passer les cuves à l’électroporateur (cliniporateur) et enregistrer chaque résultat<br><br />
<li>Incuber les cuves à 37°C pendant 30 minutes<br><br />
<li>Mettre le contenu de chaque cuve dans un tube stérile, ajouter 3ml de milieu de culture MEM 10%, puis incuber à 37°C pendant le temps nécessaire (jusqu’au test à l’annexine V) <br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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=== Détection de l’apoptose ===<br />
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La détection des cellules apoptotiques s’est effectuée par le test à l’annexine V : <br><br />
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En phase précoce de l’apoptose, on observe la translocation de la phosphatidyl-sérine à l’extérieur de la membrane plasmique. Celle-ci est mise en évidence par fixation spécifique de l'annexine V couplée à un fluorophore et analysée par cytométrie en flux. <br><br />
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<i>Matériel :</i><br><br />
<ul> <br />
<li>Iodure de propidium 1 mg/ml In vitrogen conservé au frigidaire à diluer 10 fois<br><br />
<li>Annexine V<br><br />
<li>Tampon annexine<br><br />
</ul><br />
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Travailler le plus possible dans l’obscurité (fluorophore photolabile) <br><br />
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<i>Protocole : </i><br><br />
<ol><br />
<li>Récupérer le milieu de culture (3 ml), le déposer dans un falcon 50 ml<br><br />
<li>Rincer la culture avec 3 ml de PBS, les déposer dans le falcon<br><br />
<li>Décoller les cellules à la trypsine, les déposer dans le falcon<br><br />
<li>Centrifuger<br><br />
<li>Reprendre le culot dans 0.5 ou 1 ml de PBS froid en fonction du niveau de confluence<br><br />
<li>Prélever 10 µl pour un comptage et centrifuger<br><br />
<li>Re-suspendre le culot dans du tampon annexine à la concentration de 1*106 cellule/ml<br><br />
<li>Pipetter 2 aliquots de 100 µl dans 2 tubes FACS<br><br />
<li>Ajouter dans chaque tube 5 µl d’annexine V et 1 µl de iodure de propidium<br><br />
<li>Incuber 15 min à RT<br><br />
<li>Arrêter la réaction en plaçant les tubes dans la glace fondante<br><br />
<li>Ajouter 400 µl de tampon d’annexine V<br><br />
<li>Lire au FACS le plus rapidement possible en conservant les tubes dans la glace<br><br />
</ol><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Déroulement de l’étude ==<br />
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Ne connaissant pas le temps d’expression du plasmide au sein de la lignée DU-145, nous avons réalisé un suivi cinétique de l’induction de l’apoptose en pratiquant un test à l’annexine V toutes les 6 heures pendant 48h après son électroporation. De ce fait, en couplant les taux d’apoptose de la population témoin (électroporation à vide) et de la population test (électroporation avec plasmide) avec leur taux de croissance respectifs, nous serons en mesure de déterminer l’impacte de p53wt sur l’induction de l’apoptose. La population témoin permettant d’éliminer les morts cellulaires dus à l’électroporation et au transfert de culture. <br><br />
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N’ayant pas eu un accès continu au cytomètre en flux, nous avons regroupé l’ensemble des 48h d’analyse en deux runs de cytométrie. Chaque créneau horaire de l’étude est représenté par une population cellulaire distincte. Ainsi nous avons réalisé 14 électroporations correspondant aux 7 créneaux horaires : +6h, +12h, +18h, +24h, +30h, +36h et +48h (deux par créneaux : population test + population témoin). <br><br />
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Voici le planning de répartition des électroporations: <br><br />
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[[image:planning.jpeg|center]] <br />
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Trois populations cellulaires ont donc été respectivement électroporées 12h, 24h et 36h avant le premier run de cytométrie (en rouge, à 9h, jour 3), quatre autres 6h, 18h, 30h et 48h avant le second run (en vert, à 16h, jour 3). <br><br />
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La première analyse cytométrique nous a permis d’obtenir les données pour le suivi à +12h, +24h et +36h, tandis que la seconde, nous a permis d’obtenir les données pour le suivi à +6h, 18h, +30h et +48h. <br><br />
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En couplant toutes ces données, on obtient un suivi sur 48h de l’induction de l’apoptose après électroporation de p53wt.<br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]]</span><br />
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== Résultats [1,2] ==<br />
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Chaque population cellulaire, représentant les différentes tranches horaires du suivi, a subi un test à l’annexine V à l’instant escompté. Malheureusement, une mauvaise dilution du tampon de l’annexine a causé la mort de toutes les populations cellulaires lors du test. Bien que les résultats furent probants pour les suivis à +24h, +30h et +48h par simple comparaison des populations contrôles et tests au microscope (figure 1), nous n’avons pu le confirmer par l’analyse cytométrique.<br><br />
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<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : morphologie des cellules avec ou sans incorporation de p53 wild-type</font><br><br />
</center><br />
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N’ayant pu commencer la culture des DU-145 que début octobre, les deux semaines qui nous a fallu pour atteindre la confluence nécessaire à l’expérimentation n’ont pas laissé place à la pratique d’un second essai…<br><br />
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Cependant, de nombreuses études ont montré que le fait d’amener p53 wild type au sein de cellules tumorales p53 mutées déclenchait le processus d’apoptose. C’est le cas notamment de l’étude menée par Chunlin Yang en 1995 qui a travaillé, tout comme nous, sur des cellules cancéreuses prostatiques p53 mutées (Tsu-pr1). La transfection de p53 wild type n’a pas été réalisée par électroporation mais en infectant les cellules tumorales avec des adénovirus non réplicatifs contenant p53wt (AdCMV.p53). Quarante-huit heures après avoir infecté une population tumorale avec AdCMV.p53, une forte expression de p53 est corrélée avec un taux important de mort cellulaire. Si les populations témoins (cellules non-infectées et cellules infectées avec des adénovirus contenant le gène LacZ, AdCMV.NLSßgal) montrent une morphologie tout à fait similaire et saine, une condensation et un détachement cellulaire sont observés chez la population p53 infectée. Afin de vérifier si le processus de mort suivi par ces cellules correspond bien à la voie apoptotique, une migration sur gel d’agarose de leur génome a été réalisée. <br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées (a), infectées par AdCMV.NLSßgal (b) et AdCMV.p53 (c).</font> <br><br />
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Les cellules infectées par AdCMV.p53 montrent une multitude de bandes (laddering pattern) tandis que les cellules non-infectées ou infectées par AdCMV.NLSßgal n’en montrent qu’une seul et unique de haut poids moléculaire. Ces résultats indiquent que la mort cellulaire induite par p53 wild type est d’origine apoptotique avec l’observation de la fragmentation du génome, conséquence de l’activité de la CAD (Caspase Activated DNase), une endonucléase spécifique au processus d’apoptose. <br><br />
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Un test MTT à permit de quantifier l’effet induit par l’expression de p53 wild type chez les cellules infectées. <br><br />
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[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: effet de l’AdCMV.p53 sur la survie cellulaire. Les cellules témoins et celles infectées à l’AdCMV.p53 ont été incubé dans du milieu serum-free après 1h d’infection.</font> <br><br />
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En l’absence de sérum, les cellules non-infectées et ßgal infectées continuent de proliférer. En revanche, pour les cellules p53 infectées, la prolifération est stoppée et suivie d’une importante chute de la population. Après 72h, la quasi-totalité des cellules p53 infectées sont mortes (figure 3). <br><br />
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<u><i>Selon cette étude, il apparait clairement que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit le phénomène d’apoptose et réduit de manière significative la population tumorale.</i></u><br><br />
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Des résultats similaires ont été rapportés par l’étude menée par Corrado Cirielli (en 1999) mais portant cette fois-ci sur la lignée cancéreuse U251 issue d’un gliome. Les mêmes types d’analyses que celles réalisées au cours de l’étude précédente ont été pratiquées. <br><br />
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<dt>Analyse morphologique des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c) : <br><br />
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<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphologie des cellules infectées par AdCMV.p53 (a), non-infectées (b) ou infectées par AdCMV.NULL (c), une semaine après infection. </font><br><br />
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Les populations témoins (b et c) prolifèrent et forment un tapis cellulaire une semaine après le début de l’expérience tandis que la population test (a) montrent très peu de cellules adhérentes (perte cellulaire importante) et un changement morphologique conséquent : les cellules sont sphériques.<br><br />
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<dt>Effet de l’AdCMV.p53 sur la fragmentation de l’ADN :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: électrophorèse sur gel d’agarose d’ADN isolé de cellules non-infectées, infectées par AdCMV.NULL et AdCMV.p53.</font> <br><br />
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Après infection à l’AdCMV.p53, les cellules U-251 montrent une fragmentation de leurs génomes caractéristique du processus d’apoptose.<br><br />
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<dt>Suivie de la prolifération des cellules non-infectées et des cellules infectées par AdCMV.p53 ou AdCMV.NULL par un test MTT :<br><br />
<br />
<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : prolifération des populations témoins (non-infectées ou AdCMV.NULL infectées) et de la population test par suivi de la densité optique après un test MTT.</font><br><br />
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<dd>Les cellules non-infectées et celles infectées par AdCMV.NULL prolifèrent de manière significative au cours de la semaine d’analyse tandis que les cellules infectées par AdCMV.p53 présentent une absence totale de prolifération et diminution continue de leur population.<br><br />
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<dd><u><i>Cette étude montre une nouvelle fois que le fait d’amener une version wild-type de la p53 au sein d’une population cellulaire p53 mutée induit la mort cellulaire par apoptose.</i></u><br><br />
<br />
== Conclusion [3,4,5,6,7,8,9] == <br />
<br />
Bien que nous n’ayons pu en apporter la preuve par nos propres moyens, de nombreuses études montrent qu’amener une version wild-type d’un gène suppresseur de tumeur au sein d’une cellule tumorale mutée pour ce gène permet le déclenchement de l’apoptose. Des études ''in vivo'' chez l’homme dans le cadre du cancer de la prostate, de l’ovaire et du poumon ont d’ores et déjà été menées et présentent des résultats probants. <br><br />
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La mise en place de cette étude était faite, à l’origine, pour déterminer si l’application du [[Team:SupBiotech-Paris/Introduction1Fr#drapeau|DVS]] dans la lutte anti-cancer du poumon à non petites cellules était viable ou pas. N’ayant pu conclure selon nos propres résultats, c’est l’analyse de diverses publications qui nous a permis de valider la mise en application. Selon ces publications, non seulement la mise en application est confirmée dans le cadre de notre pathologie mais peut désormais être étendue à d’autres cancers comme les carcinomes hépatocellulaires, sur lesquels le fait d’amener un gène suppresseur de tumeur déclenche également le processus d’apoptose. La seule limite étant posée par le tropisme du [[Team:SupBiotech-Paris/Concept1Fr#drapeau|vecteur tissulaire]].<br><br />
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<span style="float: right"> [[Team:SupBiotech-Paris/Ciblage_Cellulaire#drapeau|Haut de page]] </span><br />
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</html></div>Aurelhttp://2009.igem.org/Team:SupBiotech-Paris/Antitumor_actionTeam:SupBiotech-Paris/Antitumor action2009-10-21T03:55:31Z<p>Aurel: </p>
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<div>{{Template:Supbiotechcss12.css}}<br />
{{Template:SupbiotechparisEn}}<br />
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<br />
= Cell targeting =<br />
<br />
== Context ==<br />
<br />
After the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] action, comes the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]], this one is a modified bacteriophage which has the faculty to infect eukaryotic cells. Lambda phage, adapted to a concentrated presence of exogenous proteins, is a good candidate for an eukaryotic because of its high capacity of cloning and a capsid structure [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] design. The penton base originally from the adenovirus capsid appears like a promising candidate for lambda phage targeting. Indeed, it is endowed of several functions like the link to cell receptors, the internalisation of virales particles and the release of the capsid by the endosome.<br><br />
<br />
==Objective ==<br />
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Our objectives are to design a [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] of Lambda phage type recombined with a penton base from the adenovirus 5 fused at its [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]]. The [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] should be able to integer the cell, go out of the endosome, transport its DNA to the nucleus of the cell and finally to transcript its [[Team:SupBiotech-Paris/Concept3#drapeau| therapeutic genes]]. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/Antitumor_action #drapeau|Back to top]]</span><br />
<br />
== Experimental approach ==<br />
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In the framework of recombinant phage gene design we decided to fuse the adenovirus 5 penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the lambda phage. The protein D extraction from Lambda phage genome has been lead by polymerase chain reaction (PCR) with several primers couple. The same strategy has been taken for the adenovirus 5 penton base extraction from which has been extracted a plasmid coding for the virus gently offered by Dr. Karim Benihoud (UMR8121, CNRS/IGR, Villejuif, France). <br><br />
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After fusion protein formation, this one is introduced in a BioBrick plasmid. This plasmid contains a resistivity against an antibiotic for the transfection confirmation of the recombined phage into the bacteria and a reporter gene like GFP with an eukaryotic promoter, the CMV of the <i>Simian virus</i> 40 (SV40), to confirm the transfection in eukaryotic cells. This strategy permits us to prove that the bacteriophage is able to infect eukaryotic cells. <br><br />
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Unfortunately, we have not been able to build the fusion protein in time. However, scientific literature show that the [[Team:SupBiotech-Paris/Concept1#drapeau| tissue vector]] lambda phage type confection is possible by fusion of the penton base with the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] (Stefania Piersanti et al. 2004). The central sequence of the penton base, amino -acids 1 to 571, fused with the bacteriophage offer a transfection in eukaryotic cells, like the use of the RGD fragment responsible for the entry of the virus and the exit of the endosome, fragment 286 to 393. <br><br />
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<span style="float: right">[[Team:SupBiotech-Paris/ Antitumor_action #drapeau|Back to top]]</span><br />
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== Results ==<br />
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=== Design of the fusion protein ===<br />
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For the design of the fusion protein, we decided to extract separately the penton base to the [[Team:SupBiotech-Paris/Biobricks#drapeau| D protein]] of the du lambda phage thanks to primers containing a BalI restriction site on the de la [[Team:SupBiotech-Paris/Biobricks#drapeau| protein D]] reverse primer and the penton base forward primer. Moreover the finale fusion protein contains specific BioBricks fragments to its ends. <br><br />
For these 2 genes extraction we used the following primers: <br><br />
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First and second pair’s genes extraction: <br><br />
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D protein of the Lambda phage: <br><br />
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Forward : ATG-ACG-AGC-AAA-GAA-ACC-TT; <br><br />
Reverse : AAA-AAA-ATC-CCG-TAA-AAA-AAG-C. <br><br />
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Adénovirus 5 penton Base : <br><br />
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Forward : AAT-GGC-CAA-TGC-GGC-GCG-CGG-CGA-TG <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA-TCA-AAA-AGT-GCG-G <br><br />
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Third pair for extension of the BalI restriction site and the BioBrick prefix only for the [[Team:SupBiotech-Paris/Biobricks#drapeau|D protein]] (already done for the penton base). <br><br />
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Forward : CGA-AAA-AAA-TGC-CCT-AAA-AAA-AAC-CGG-T <br><br />
Reverse : AAT-GGC-CAA-AAA-AAA-TCC-CGT-AAA-AAA-AGC <br><br />
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Fourth pair for the D protein fusion amplification after ligation of the two fragments. <br><br />
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Forward : CTT-AAG-CGC-CGG-CGA-AGA-TC <br><br />
Reverse : CTG-CAG-CGG-CCG-CTA-CTA-GTA <br><br><br />
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PCR results are presented in figure X. We check that there is the right amplification size fragment 1715bp for the penton base of the sample number 9 and 385bp for the D protein in samples 5 and 6. However there is lots of mismatching during amplification cycles. This can have a negative effect on the result of the final amplification. <br><br />
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[[image:M2109.png|center]]<br />
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<i>Figure 1: PCR of D protein BioBricks (1, 2, 3) and the penton base (4, 5, 6), D protein (5 and 6) and penton base (7, 8, 9) with BalI sites </i><br><br />
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<span style=«float: right»>[[Team:SupBiotech-Paris/ Antitumor_action #drapeau|Back to top]]</span><br />
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=== Transfection of eukaryotic cells by the lambda phage recombined with the penton base fused to the D protein (Stefania Piersanti et al., 2004) ===<br />
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A cytofluorimetric study has been done to analyze the transfection rate of recombined lambda phages. Figure X show cytofluorimetric results of COS-1 cells analyze after to have been exposed to a concentration of 10^6 PFU/cells of recombinants phages, Pb (1-571) or Pb (286-393).<br />
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[[image:VT1.png|center]]<br />
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[[image:VT2.png|center]]<br />
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<i> Figure 2 : Analyze of the GFP fluorescence on non recombined lambda phages (Lambda), recombined lambda phages with the fragment 286-393 of the penton base (LambdaPb286-393), recombined lambda phages with the complete penton base (1-571), GFP tagged adenovirus (Ad10 and Ad100)</i><br><br />
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Firstly, we observe that the recombined phage show a tag difference independently of the fragment of the penton base used compared to the non transformed bacteriophage. Secondly, the recombined phage with the RGD fragment alone (286-393) has a higher fluorescence than the phage with a complete fragment and closer to the adenovirus one (figure X). <br><br />
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<span style=«float: right»>[[Team:SupBiotech-Paris/ Antitumor_action #drapeau|Back to top]]</span><br />
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== Discussion ==<br />
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Even if the tissue vector has not been finished, the scientific literature shows that a recombinant phage creation with a protein coding the adenovirus penton base is possible. It demonstrated as well that fragments coding for RGD sequences alone have a higher capacity to infect eukaryotic cells compared to the penton base complete fragment de la (figure 2). In the case of our application it is possible to use a recombined lambda phage to insert our therapeutic gene. <br><br />
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<span style=«float: right»>[[Team:SupBiotech-Paris/ Antitumor_action #drapeau|Back to top]]</span><br />
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== Conclusion ==<br />
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To conclude the RGD fragment of the penton base alone has a higher efficiency of interaction with integrines of eukaryotic cells, however for our project it was more judicious to use the complete sequence of the penton base (fragment 1-571) because with the use of the [[Team:SupBiotech-Paris/Concept2#drapeau| cell vector]] and the induction system by doxycycline give an injection very fast and targeted of our bacteriophages. The use of a highly efficient transfection system is not advised because phages have not the time to disperse properly and will infect several times the same cell. The use of the complete fragment of the penton base is sufficient for the phage to infect properly eukaryotic cells and let it time to have a bigger dispersion. <br><br />
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<span style=«float: right»>[[Team:SupBiotech-Paris/ Antitumor_action #drapeau|Back to top]]</span><br />
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= Application =<br />
== Context ==<br />
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In non-small cell lung cancer, or NSCLC, like in all other cancers, the loss of apoptotic capacity of tumor cells is due to the functional loss of various tumor suppressors incoming in the apoptotic pathway.<br><br />
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The [[Team:SupBiotech-Paris/Introduction1En#drapeau|DVS]] application in the anticancer fight is based on the reactivation of this apoptotic pathway by bringing in tumor cells wild type genes coding for non-functional tumor suppressors.<br><br />
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The [http://www.sanger.ac.uk/genetics/CGP/cosmic/ COSMIC project] from [http://www.sanger.ac.uk/ Sanger institute] permits us to determine which genes to bring to the [[Team:SupBiotech-Paris/Concept3En#drapeau|therapeutic plasmid]] in the non-small cell lung cancer case. This project sums up all detected mutations for each type of cancer in function of their appearance frequency. So, from their data, the loss of apoptotic capacity of tumor cells for lung cancer can be due to the functional loss of proteins from the following genes :<br><br />
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[[image: gènes mutés.jpeg]]<br />
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These different genes, that play a predominant role in the application of the apoptotic process and which are the most susceptible to be mutated in the lung cancer case, compose the [[Team:SupBiotech-Paris/Concept3En#drapeau|therapeutic plasmid]].<br><br />
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== The objective ==<br />
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The objective of this study is to check that if a wild type version of a tumor suppressor gene inside the tumor cell for which the own version is mutated, induce or not the apoptotic phenomenon.<br><br />
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== Experimental approach ==<br />
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=== Cancer cell line and reported gene ===<br />
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We select between several cell lines we had at disposal, a cancer cell line which the cancer origin is due to the mutation of a tumor suppressor gene. We possess the wild type of TP53 gene, the prostatic cancer p53 mutated DU-145 line retains our attention. <br><br />
So, we will test if bringing a wild type version of the p53 protein (p53wt) in the DU-145 cell line permits to induce an apoptotic process.<br><br />
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<i>Cell culture protocol : </i><br><br />
<ol><br />
<li>Take out ampoule from liquid nitrogen<br><br />
<li>Place the ampoule in 37°C water bath for 5 minutes<br><br />
<li>In a 50 ml Falcon tube, put 9 ml of 10% MEM + 1 ml of ampoule<br><br />
<li>Harvest 5 min at 1200 rpm<br><br />
<li>Discard the supernatant without touching pellet cells (DMSO elimination) <br><br />
<li>Resuspend pellet in 1 ml of media<br><br />
<li>Put the suspension in a new T25 containing 5 ml of media<br><br />
<li>Incubate at 37°C<br><br />
<li>Do not forget to change the media the day after to eliminate all DMSO traces <br><br />
<li>One week later, cells are at 100% confluence<br><br />
</ol><br />
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=== TP53 gene incorporation ===<br />
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Incorporation of the plasmid containing p53wt, pcDNA3 CMV+p53wt, insideDU-145 cells is done by electroporation. <br><br />
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<i>Material :</i> <br><br />
<ul><br />
<li> DU-145 cells <br><br />
<li>pcDNA3 CMV+p53wt plasmid<br><br />
<li> Electrocompetent culture media<br><br />
<li>Trypsin<br><br />
<li>PBS<br><br />
<li>Icebox<br />
<li>Electrotransfer Cuvette <br />
<li>Centrifuge<br />
<li>Incubator<br />
<li>Electroporator (cliniporator)<br />
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<i>Protocol: </i> <br><br />
<ol><br />
<li>Discard the media of T25 containing DU-145<br><br />
<li>Rinse with PBS<br><br />
<li>Add 500 µl of trypsin and let it acts for 3 minutes at room temperature <br><br />
<li>Add 5 ml of 10% MEM to neutralize trypsin<br><br />
<li>Suspend cells<br><br />
<li>Recover media containing DU-145 in a tube and harvest at 1000rpm for 10 minutes<br><br />
<li> Discard the supernatant and resuspend the pellet in Xµl (X= 90µl x Number of cuvettes) of electrocompetent media (around 5x105 cells per cuvettes) <br><br />
<li>Suspend your DNA solution in electrocompetent media (18x10-2g/L) <br><br />
<li>Add 10µl DNA solution per cuvette<br><br />
<li> Add 90µl of the cell suspension <br><br />
<li>Put cuvettes in ice<br><br />
<li>Pass cuvettes to the electroporator (cliniporator) and save each result <br><br />
<li>Incubate cuvettes at 37°C for 30 minutes<br><br />
<li>Put the content of each cuvette in a sterile tube, add 3ml of MEM 10% culture media, and incubate at 37°C for the necessitate time (until the annexin V assay) <br><br />
</ol><br />
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=== Apoptosis detection ===<br />
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Detection of apoptotic cells is done by the annexin V assay: <br><br />
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In the early stage of the apoptosis, we observe the phosphatidyl-serine translocation outside the cell membrane. These is highlighted by the specific fixation of the annexin V coupled with a fluorophore and analysed by flow cytometry. <br><br />
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<i>Material :</i><br><br />
<ul> <br />
<li> Propidium iodide 1 mg/ml Invitrogen stored cold in the fridge, diluted 10 times<br><br />
<li>Annexin V<br><br />
<li>Annexin buffer<br><br />
</ul><br />
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Work as much as possible in the dark (fluorophores are photolabile) <br><br />
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<i>Protocol : </i><br><br />
<ol><br />
<li>Recover culture media (3 ml), put it in a Falcon tube of 50 ml<br><br />
<li>Rinse the culture with 3 ml of PBS, and dispose it in the Falcon tube<br><br />
<li>Remove cells with trypsin, and dispose them in the Falcon tube<br><br />
<li>Harvest<br><br />
<li>Resuspend the pellet in 0.5 or 1 ml of cold PBS in function of the confluence level<br><br />
<li>Take 10 µl to count and harvest<br><br />
<li>Re-suspend the pellet in annexin buffer at a concentration of 1*106 cell/ml<br><br />
<li>Take 2 aliquots of 100 µl in 2 FACS tubes <br><br />
<li>Add in each tube 5 µl of annexin V and 1 µl of propidium iodide<br><br />
<li>Incubate 15 min at RT<br><br />
<li>Stop the reaction by put tubes in melting ice <br><br />
<li>Add 400 µl of annexin V buffer<br><br />
<li>Read in FACS as quick as possible and let tubes in the ice<br><br />
</ol><br />
<br />
== The sequence of studies ==<br />
<br />
The time of the plasmid expression in DU-145 cell line were not known so, we realized a kinetic monitoring of the apoptosis induction by making an annexin V assay every 6 hours for 48h after its electroporation. By the way, by coupling apoptosis rate of the population control (blank electroporation) and the population assay (electroporation with plasmid) with their respective growth rate, we will be able to determine the p53wt impact on apoptosis induction. The population control permits to eliminate cell death due to electroporation and to the culture transfer. <br><br />
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Because we had not a continuous access to the cytometer, we grouped the all 48h analyses in 2 cytometry runs. Each time slot of the study is represented by a distinct cell population. So, we realized 14 electroporations corresponding to the 7 time slots: +6h, +12h, +18h, +24h, +30h, +36h and +48h (two by slot: population assay+ population control). <br><br />
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Here is the allocation planning of electroporations: <br><br />
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[[image:planning.jpeg]] <br />
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Three cell populations were respectively electropored 12h, 24h et 36h before the first cytometry run (in red, at 9h, day 3), four others 6h, 18h, 30h and 48h before the second run (in green, at 16h, day 3). <br><br />
<br />
The first cytometric analyze permits us to obtain data for the monitoring at +12h, +24h and +36h, while the second one, permits us to obtain data for the monitoring at +6h, 18h, +30h and +48h. <br><br />
<br />
By coupling all these data, we obtain a monitoring on 48h of the apoptosis induction after p53wt electroporation. <br><br />
<br />
== Results ==<br />
<br />
Each cell population, which represents different time range of the monitoring, has been subjected to an annexin V assay at the instant looked for. Unfortunately, a wrong dilution of the annexin buffer caused the death of each cell populations during the test. Even if results were convincing for the monitoring at +24h, +30h and +48h by simple comparison between the control and the test population in the microscope (figure 1), we could not confirm it by cytometric analyse. <br><br />
<br />
<center><br />
[[image:figure 1bis.jpeg]]<br><br />
<font size="1"><i>Figure 1</i> : cells morphology with or without p53 wild-type incorporation </font><br><br />
</center><br />
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Because we could only start DU-145 culture at the beginning of October, the two weeks needed to reach the necessary confluence did not let the place to a second chance… <br><br />
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However, several studies showed that the fact to bring p53 wild type into tumor mutated cells launched the apoptosis process. It is notably the case of the study leaded by Chunlin Yang in 1995, who were working, like us, on mutated p53 prostatic cancer cells (Tsu-pr1). The p53 wild type transfection were not realized by electroporation but by infecting tumor cells by non replicatives adenoviruses containing p53wt (AdCMV.p53). 48 hours after to have infected a tumor population with AdCMV.p53, a high expression of p53 is correlated with an important rate of cell death. If control populations (non-infected cells and cells infected with adenovirus containing lacZ gene, AdCMV.NLSßgal) show a similar and healthy morphology, condensation and cell detachment are observed in p53 infected population. To check if the death process followed by cells correspond to the apoptotic way, a migration on agaraose gel of their genome were realized.<br><br />
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[[image:figure 2bis.jpeg|float|left]]<br><br />
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<font size="1"><i>Figure 2 </i>: Electrophoresis on agar gel of isolated non-infected DNA cells (a), infected by AdCMV.NLSßgal (b) and AdCMV.p53 (c).</font> <br><br />
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Infected by AdCMV.p53 cells show multiple bands (laddering pattern) while non-infected cells or AdCMV.NLSßgal infected cells show a unique one at high molecular weight. These results indicate that the cell induced by p53 wild type is from apoptotic origin with the observation of the genome division, consequence of the CAD (Caspase Activated DNase) activity, a specific endonuclease to the apoptoctic process. <br><br />
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A MTT test permitted to quantify the effect induced by the p53 wild type expression into infected cells.<br><br />
<br />
[[image:figure3bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 3 </i>: AdCMV.p53 effect on cell survive. Control and AdCMV.p53 infected cells were incubated in serum-free media after 1h of infection.</font> <br><br />
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In serum absence, non-infected and ßgal infected cells continue to proliferate. In contrast, for p53 infected cells, proliferation is stopped and followed by an important fall of population. After 72h, nearly the totality of p53 infected cells are dead (figure 3). <br><br />
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<u><i>According to this study, it appears clearly that the fact to bring a p53 wild-type version into the p53 mutated cell population induce the apoptosis phenomenon and decrease significantly the tumor population.</i></u><br><br />
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Similar results were reported by the study leaded by Corrado Cirielli (in 1999) but this time on the U251 cancer strain from a glioma. Same types of analyses than these realized during the previous study were done. <br><br />
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<dt> Morphologic analyse of AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c) : <br><br />
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<dd>[[image:figure4bis.jpeg]]<br> <br />
<font size="1"><i>Figure 4</i> : morphology AdCMV.p53 infected cells (a), non-infected (b) or infected by AdCMV.NULL (c), after one week infection. </font><br><br />
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Control populations (b and c) proliferate and form a cell layer one week after the beginning of experiences while the control population (a) show very few adherent cells (important cell loss) and a consequent morphologic change: cells are spherical.<br><br />
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<dt> AdCMV.p53 effect on DNA division :<br><br />
<dd>[[image:figrue5bis.jpeg|float|right]]<br><br />
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<font size="1"><i>Figure 5 </i>: electrophoresis on agar gel of isolated DNA of non-infected cells, infected by AdCMV.NULL and AdCMV.p53.</font> <br><br />
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After AdCMV.p53 infection, U-251 cells show a division of their genome characteristic of the apoptosis process.<br><br />
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<dt>Monitoring of the non-infected cells and infected by AdCMV.p53 or AdCMV.NULL cells by a MTT test:<br><br />
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<dd><center>[[image:figure6bis.jpeg]]</center><br> <br />
<font size="1"><i>Figure 6</i> : Control population proliferation (non-infected or AdCMV.NULL infected) and the test population by monitoring of the optical density after a MTT test.</font><br><br />
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<dd> Non-infected cells and AdCMV.NULL infected cells proliferate in a significant way during the week of analysis while AdCMV.p53 infected cells present a total absence of proliferation and a continuous decrease of their population.<br><br />
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<dd><u><i>This study show one more time that the fact to bring a p53wild-type version into a mutated p53 cell population induce cell death by apoptosis.</i></u><br><br />
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== Conclusion ==<br />
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