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 Culture of feeder-free ES cells- CGR8, E14tg2a and their derivatives Sodium pyruvate : Gibco 11360-070, 100mM solution, 100mlPorcine skin : gelatin Sigma G9391, type B or G2625, typeAPhosphate buffered saline : Sigma P4417, 100 tabletsTrypsin-EDTA (0.25% trypsin, 1mM EDTA) : Gibco 25200-056/072DMSO2-mercaptoethanol(2-ME) : Wako etcLeukemia inhibitory factor (LIF) ESGRO (GibcoBRL) or prepared by transient transfection the expression vector pCAGGS-LIF in COS-7/BMT-10 cells.
should be tested by plating efficiency and colony morphology Geneticin (G418) : Sigma's one is cheapest!Hygromycin B (Hyg): GibcoBRL 10687-010 50 mg/ml in PBSBlasticidin S hydrochroride (BlaS): FUNAKOSHI KK-400 mgZeocin : Invitrogen 100mg/ml solutionpuromycin : Sigma P7255Gancyclovir (Ganc): Roche/Tanabe Denosine 500 mgHAT : Sigma H0262 HAT MEDIA SUPPLEMENT 50x, 10vials : 129/Ola-derived wild-type ES cells.
: 129/Ola-derived HPRT-negative ES cells. HPRT-minigene can be used as an additional option of selectable marker by combination with HAT selection.
MG 1.19 : CCE ES cells carrying the primary episomal vector pMGD20neo for supertransfection.
: CGR8 ES cells carrying IRES-hytk(hygromycin resistant+HSV-tk fusion) in one of Oct-3/4 locus, which allow selection or elimination of Oct-3/4-positive stem cells.
:E14tg2a ES cells carrying IRES-BSD in one of Oct-3/4 locus, which allow selection of Oct- positive undifferentiated stem cells.
: CGR8-derived ES cells carrying tetracycline-regulatable Oct-3/4 transgene and IRES-zeocin casette in one of Oct-3/4 locus, which allow selection or elimination of Oct-3/4-positive stem cells. These cells should be maintained in the presence of Tc to k e e pstem-cell-renewal.
: ZHTc6-derived ES cells maintained by tetracycline-regulatable Oct-3/4 transgene. Both of Oct-3/4 locus are disrupted by IRES-zeocin and IRES-BSD KO vectors. These cells should be maintained without Tc to keep stem-cell-renewal.
GMEM10% FCS1xNEAA1mM sodium pyruvate10-4M 2-ME1000 U/ml LIF prepare 10- 1M stock solution (1000X) by adding 0.1ml 2-ME to 14.1 ml PBS. Filtrate with 0.2µ filter and store upto4 weeks at 4C.
Gelatin solution : 0.1% gelatin in PBS. Sterilized by autoclave and G418 : 100-200mg(activity)/ml in 1M HEPES buffer. Filtrate BlaS : 10mg/ml in dH2O. Filtrate (0.20micron) and store at -20oC.
puromycin : 2mg/ml in dH2O. Filtrate (0.20micron) and store at Ganc : Dissolve 500mg in 10ml dH2O to prepare 0.2M stocksolution. Store at -80oC. Dilute the stock solution 1 in 2000dH2O to final concentration of 1mM (1000x solution) and store zeocin : Dilute the original solution 10 times with dH2O to prepare All dishes, flasks and plates should be gelatinized before use by themethod described below : 1. Add gelatin solution2. Keep 10 min at room temperature.
3. Aspirate thoroughly.
1. Transfer the freeze stock vial to 37oC water bath.
2. Incubate it till the ice start to thaw.
3. Transfer cell suspension(0.5ml) into a centrifuge tube.
4. Add 5ml ES medium gradually.
5. Spin down the cells : 1500rpm, 5min.
6. Suspend cells in 20ml of mediumand seed in 75cm2 flask or 90mm dish x2.
7. Change medium in the next day.
(usually 3 days after seeding or 2 days when cells cover 80% of thesurface) In the case of 90mm dish- You can correct 5x106 cells per dish.
1. Aspirate medium and wash cells with PBS twice.
2. Add 0.3ml trypsin-EDTA3. Incubate at 37oC for few min.
4. Add 3ml of ES medium5. Count the number of cells.
6. Spin down the cells 7. Suspend cells in ES medium.
8. Seed 1.3-1.5x105 cells / cm2 25cm2 small flask : 3-4x105/8ml medium75cm2 middle flask : 1x106/20ml medium150cm2 large flask : 2x106/40ml medium60 mm dish : 1x105/4ml medium90 mm dish : 5x105/10ml medium 9. Change medium (day 2)10. Passage (day 3) Numbers of cells usually reach 10-times of seeded ones.
1. Harvest cells as (B), suspend 2-3x106 cells/0.25 ml ES medium 2. Prepare 2x freeze stock solution (20% DMSO in ES medium) and 3. Add equal volume of 2x stock solution to cell suspension with 4. Transfer cell suspension to freeze vials (0.5 ml/vial).
5. Keep the vials at -80oC for overnight.
6. For long-term storage, keep them in the gas phase (caution! : not in liquid phase) of liquid N2 tank.
Ethanol pretipitation pellets of linealized vector (100 µg/electro-poration) should be dissolved in 50 µl of dH2O and keep at 4oC forovernight. Then add 350 µl of PBS.
1. Harvest 2-4x107 cells for 1 electroporation.
2. Wash cells with PBS twice.
3. Count the cell number and suspend them in 400 µl of PBS.
4. Add DNA solution to the cell suspension.
5. Keep 10-20 min. on ice.
6. Transfer suspension to the electroporation cubette (0.4cm 7. Give pulse with Bio-Rad Gene Pulser set at 0.8kV/3µF. The resulting time-constance is usually 0.1 or 0.2.
8. Transfer pulsed cells to the tube containing ES medium and 9. Seed 2x106 cells / 90mm plate with 10 ml of ES medium.
10. Next day, change the medium to fresh one with appropriate You will see -300 (with promoter) or -30 (promoterless) colonies /plate 7-10 days after electroporation.
For supertransfection of episomal vectors into MG1.19 or itsderivatives, please use 5x106 cells / electroporation with 10-20 µgcircular DNA and give pulse at 0.2kV/960µF. The resulting time-constance is usually 15.0-20.0. You will see 1-5% of treated cellsas stable transfectant colonies.
1. Aspirate the medium and wash the plate with PBS.
2. Add 5 ml of PBS.
3. Add 10 µl of trypsin-EDTA per well in 96 well plate.
4. Pick up colonies with pipetteman and put them into trypsin- 5. Add 100 µl of ES medium and suspend the colonies by gentle 6. Transfer cell suspension to 48 well plate containing 500 µl of ES 7. Pass to 2x24 well plate 3-4days after picking up.
(One for freeze stock and another for DNA preparation) (F) Short-term freezing of ES cell clones in 24-well plates Please refer Protocol 13 & 14 in Joyner's textbook p60.
Please refer Protocol 12 in Joyner's textbook p58.
Appendix. Concentration of drugs for selection Smith, A.G. : Culture and differentiation of embryonic stem cells. J.
Tiss. Cult. Meth., 13, 89-94, 1991.
Joyner, A.L. : Gene Targeting - a practical approach. IRL PRESS.

Source: http://www.cdb.riken.jp/pcs/protocol/ESprotocol.pdf

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