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