Stable Cell Lines

Contents

7-24-2009

• start new cell culture: HeLa, U2-OS

7-27-2009

• start new cell culture: MCF7
• split HeLa and U2-OS (both from 7-24-09) 1:10

7-28-2009

• start with stable transfection of HeLa, MCF7, U20S cells with pFRT/lacZeo following Effectene protocol
• Washed(PBS) and changed medium of all cell lines.
• Splitting HELA- and U2-OS (from 7-27-2009) 1:10

7-30-2009

• removed old medium
• added fresh medium with Zeocin (stock at 100 mM (used all the stock for 50 ml medium) --> add to medium to get a concentration of 200 µM (1:500))

8-03-2009

• stable transfection: changed medium (without washing)
  • Hela --> quite a few dead, but some areas seem to be dense
  • MCF7 --> many dead cells, but dense nonetheless
  • U2OS --> fairly dense and not so many dead cells

8-04-2009

• splitted MCF7 and U2OS with Zeocin medium in new 6-well plate (each 1:10 and 1:5)

8-06-2009

• changed zeocine medium of HELA, MCF7 (original well, 1:10, 1:5) and U2OS (original well, 1:10, 1:5)

8-07-2009

• Hela: original 6-well --> kept 1/3, split rest in 2 petri dishes with 3 ml medium with Zeocin (--> final volume of 3,7 ml)
• MCF7/U2OS: used 1:5 dilution (from 8-4-2009) --> resuspended in 2 ml and added 1 ml to 3 ml medium with Zeocin in two petri dishes

(MCF7 looked strange --> very long, almost like fibers)

8-10-2009

• Zeocin selection: picked 4 Hela and 4 U2OS foci from petri dishes and transfered them to 24-well (see methods);
• MCF-7 not ready
• changed Zeocin medium in 6-wells (Hela, 1:10 MCF7, original MCF7, 1:10 U2OS -> original U2OS too dense)
• used second petri dishes (Hela, U2OS) to split in 96-well, so that there should be 1 cell per well -> U2OS: counted 3x10⁴ cells/ml, diluted 1:2, used 0,6 µl per well and prepared 24 wells; Hela: counted about 0,75-1x10⁴ cells/ml, used 0,9 µl per well and prepared 24 wells

8-13-2009

• selection of stable clones of U2OS
  • single cell wells: 5 96-well plates with one cell per well in 150 µl Zeocin DMEM+++ (originally 22,5*10^4 cells/ml)
• selection of stable clones of HeLa
  • 7 cloning disks placed on colonies visible without microscope (following protocol, see methods)
  • transfered cloning disk to 96-well

8-18-2009

• split U2-OS from 6-well in large petri dishes (1:30, 1:50)
• HeLa and MCF-7 from 6-well discarded because they looked sick

8-21-2009

• HeLA from 96-well (8-13-09) trypsinized (20 µl Trypsin, 5 min) and filled up with 200 µl DMEM/zeocin
  • should spread the cells over the entire well

8-23-2009

• restarted with Hela and MCF-7
  • split in 6-wells (4 wells, 10^5 per well)

8-24-2009

• picked 7 U2-OS foci from 1:30 petri dish in 96-wells
• transfected Hela in 6-wells (from 8-23) with lacZeo/FRT
• MCF-7 not dense enough for transfection

8-25-2009

• transfected MCF-7 in 6-wells (from 8-23) with lacZeo/FRT
• changed Medium (DMEM+++) of transfected HeLa (from 8-24-09)

8-26-2009

• changed Medium of transfected HeLa (from 8-24-09): now kept under selectionpressure with Zeocin in DMEM+++ (1:1000)
• transferred HeLa cells derived from one clone from 96-well (13-08-09) to 6-well in order to expand them
• removed transfectioncomplexes from MCF-7 (8-25-09)by changing medium (DMEM+++)

8-27-2009

• split transfected MCF-7 (from 8-25-09) 1:2 and started keeping them under selectionpressure with Zeocin in DMEM+++ (1:1000)

8-28-2009

• trypsinized HeLa and MCF-7 ( both in 6-well) and resuspended them in 2 ml DMEM+++/Zeocin
• transferred 1 ml of cell suspension to 7 ml DMEM+++/Zeocin in petridish (10)
• changed medium (DMEM+++/Zeo) of HeLa in 6-well (hopefully stable clon)

9-02-2009

• splitted MCF-7 from 6-well 1:2 in another 6-well
• picked another 8 foci from U2OS (petri dish) into 96-wells with DMEM+++/Zeocin
• splitted rest of U2OS from petri dish 1:3 in new petri dish (10)
• found cells in 96-wells with U2OS (2-3 cells/well)

9-04-2009

• transferred 10 wells of U2-OS (2-3 cells/well) from 96-wells into 24-wells

9-07-2009

• transferred the 10 wells of U2-OS (2-3 cells/well) from 24-wells into 6-wells

9-08-2009

• splitted "stable" Helas from upper 6-well in flask (25cm²)

9-10-2009

• splitted "stable" Helas from upper 6-well 1:3 into another flask (25cm²) (one third stayed in old 6-well, one third into flask and one third for cotransfection (3 times 3x10^4 in 6-wells))
• washed and trypsinized "stable" Helas from lower 6-well
• trypsinized U2-OS (picked with cloning disks 9-02-2009) and left in 96-well
• washed and trypsinized Helas and MCF-7 (under Zeocin pressure, not picked yet)
• spotted very weird/huge focus of Hela in one of those wells

9-11-2009

• prepared two 24-wells with MCF-7 for Lipofectamin testing and transfection with linearized lacZeo plasmid
• splitted four confluent colonies (#1,3,5,7) of "stable" U2-OS from 6-well (9-7) 1:3 into two other 6-wells for co-transfection with recombinase

9-12-2009

• transfected (Lipofectamin) MCF-7 in 24-well with linearized lacZeo plasmid (SacI digested)
• prepared one 24-well with Helas for Lipofectamin testing
• "stable" Helas for co-transfection in 6-wells not dense enough --> wait before transfecting
• "stable" U2-OS in 6-wells for co-transfection confluent --> splitted 1:3 (2/3 in 25 cm² flasks)

9-13-2009

• changed medium of 24-well with linearized lacZeo --> added Zeocin for selection of stables
• transfected Helas with Lipofectamin

9-14-2009

• splitted "stable" Helas from first flask 1:2 into large flask (75cm²)
• co-transfected "stable" Helas in 6-wells (with Lipofectamin):
  • p55, p43, p45 (CMV, Cherry, Recombinase)
  • p31, p45 (JeT, Recombinase)
  • p55, p45 (CMV, Recombinase)
• co-transfected four (#1,3,5,7) "stable" U2-OS (from 96-well with 2-3 cells/well) with p55, p43 & p45 (CMV, Cherry, Recombinase)
• transferred three U2-OS colonies (#1,2,3 from cloning disks) into 24-wells

9-15-2009

• changed medium on "stable" co-transfections --> DMEM+++ +Zeocin in the morning
• changed medium on "stable" co-transfections --> DMEM+++ +Hygromycin (1:200) before leaving
• prepared 1x10^5 cells (stable HeLas) for genomic DNA extraction --> works (15 ng/µl)

9-16-2009

• PCR of FRT-site (to check if it's actually there) with O.243&O.244 and genomic DNA

9-17-2009

• split "stable" co-transfected HeLa 1:2 into another 6-well
• split "stable" co-transfected U2-OS 1:5 into another 6-well (except for #5 --> wash and put new medium on; leave in 6-well)
• put PCR of FRT-site (9-16) on gel --> looks good (fragment should be 240 bp; is between 200 and 300 bp)

9-19-2009

• changed zeocin medium of stable U2-OS (1,3,6,7 in flask)
• split stable HeLas 1:10 (flask)

9-21-2009

• stable US-OS "2-3 cells/well" split 1:5
• stable US.OS "cloning disk" transferred to 6 well

9-22-2009

• isolation of genomic DNA from
  • stable US-OS "2-3 cells/well" # 1,3,6,7
  • stable U2-OS "cloning disk" 24 well
  • stable HeLa
  • stable MCF-7 (Yara)

very bad yield, needs to be redone

9-24-2009

• split stable US-OS "2-3 cells/well" # 1,3,6,7 from 6 well 1:2 in other 6 wells
• prepared 6 6 wells with 10^5 stable Hela cells for transfection
• transferred stable U2-OS "cloning disk" :
  • from 96 well to 6 well
  • # 5,6,7 from 6 well to flask

9-25-2009

• cotransfected (each 3 6 wells) stable Helas and U2OS in 6 wells with recombinase plasmid (p45) and
  • CMV_mCherry(p55)
  • Jet_mCherrry (p31)
  • JetProx_CmvCore_mCherry(p48)
• cotransfected only stable Helas in 6 wells with recombinase plasmid (p45) and
  • constitutive S4
  • constitutive L4

9-26-2009

• changed medium of yesterday's transfections
• checked flourescnece of transfected cells:
  • CMV very bright
  • Jet, CMV_Jet , constituiv S4 and L4 no flourecence visible

9-27-2009

• checked transfections again: Jet, CMV_Jet , constituiv S4 and L4 flourescence also visible
• changed medium of the cells: DMEM+++/Hygromycin (200 µg/ml)

9-28-2009

• cells of transfections are too dense; according to manual Hygromycin not effective if more than 25% confluent
  • split them 1:2 in petridishes
• isolation of genomic DNA from
  • U2-OS "2-3 cells/well" # 1,3,6,7
  • U2-OS "cloning disk" # 5,6,7
  • HeLa (two samples of same clone)
  • MCF-7 ( from Yara,two samples of same clone)

9-29-2009

• froze stable cell lines (3 of U2-OS, 1 of HeLa, 1 of MCF-7 -> all from picked foci using cloning discs)
• checked "surviving rate" of cells under hygromycine pressure
  • HeLa: look pretty bad, only very few surviving
  • U2-OS: look better, surviving ones express GFP
• started LAM-PCR according to protocol Schmidt et al. 2007
  • stopped with overnight capture after second linear PCR

Schmidt M., Schwarzwaelder K.,Bartholomae C., Zaoui K., Ball B.,Pilz I.,Braun S.,Glimm H.,von Kalle C., (2005) High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR) Nature Methods 4:12, 1051-1057

9-30-2009

• changed Hygromycine medium on transfections (remaining 6-wells and petri dishes; 200 µg/ml)
• continued with LAM-PCR
  • began with hexanucleotide priming and stopped with overnight capture after first exponential PCR (Primer LCI)

10-01-2009

• continued with LAM-PCR
  • second exponential PCR with primer LCII and reaction specific primer
  • gelelectrophoresis on 2 % argarose gels
  • reaction 9 and 10 successful, but more then one integration site per cell line
  • need to do topovector cloning, hopefully possible via GATC

10-02-2009

• changed hygromycine medium: diluted 1:500 -> 100 µg/ml (stables should be selected -> make them grow faster with lower antibiotic concentration)

10-05-2009

• new Hygromycine medium (100 µg/ml)

10-08-2009

• picked foci of stable integrations (transfected 9-25) in 96-wells using cloning disks:
  • CMV (U2-OS): 5 foci
  • S4 (HeLa): 2 foci
  • JeT (HeLa): 1 focus
  • L4 (HeLa): 1 focus

• used regular DMEM+++ (w/o Hygromycine) overnight
• also put regular DMEM+++ on petri dishes overnight

After this day, we only changed medium on the plates with the foci. That is, because we had the LAM-PCR results that showed we had multiple integration sides. Thus, we figured we should not invest the time to clone the mixture of bands into topo vectors in order to sequence them.