Careful maintenance of ES cells is crucial for successful germline transmission of gene trap cell lines. We generally change the medium every day, and do not allow the cells to grow to confluence. Several previously published protocols describe procedures for maintaining ES cells on fibroblast feeder layers (15-18). The conditions outlined below apply to the maintenance of a feeder-independent line of ES cells, CGR8, which rely on an exogenous source of DIA (also known as leukocyte inhibitory factor [LIF]). These cells are karyo-typically male and were derived from the 129/Ola strain of mice as described (19). Feeder-independent cell lines are more convenient to work with and contribute as efficiently to the germline of mice as feeder-dependent cell lines. We find that 80% of our gene trap cell lines contribute to the germline at an average rate of 1 germline male/10 C57Bl/6 blastocysts injected (Skarnes, unpublished results).
All ES cell manipulations should be carried out in a laminar flow hood. All solutions should be warmed in a 37°C waterbath prior to use.
3.1.1. Thawing ES Cells
1. Coat a 25-cm2 tissue-culture flask with 0.1% gelatin and aspirate off.
2. Quickly thaw ES cells (one-half of a confluent 25-cm2 flask or approx 5 x 106 cells) in a 37°C water bath, and transfer them to a disposable centrifuge tube containing 10 mL of prewarmed medium.
3. Spin the cells down in a bench-top centrifuge at 260g for 3 min.
4. Aspirate medium off and gently resuspend cells in 10 mL of pre-warmed medium.
5. Transfer to 25-cm2 tissue-culture flask and grow in a humidified 37°C/6% CO2 incubator.
6. Change medium after about 8 h of growth to remove dead cells and any remaining DMSO (an inducer of ES cell differentiation).
7. Medium should be changed every day.
1. ES cells are passed once the have nearly reached confluence. For a 25-cm2 flask, aspirate medium off, and add 5-10 mL of PBS down the opposite side of the flask to where the cells are growing. Rock the flask gently and aspirate off. Repeat.
2. Cover cells with 1 mL of trypsin, and incubate at 37°C for 1-2 min. Incubate for longer if the cells are not in a uniform suspension.
3. Add 9 mL of medium to stop trypsinization.
4. Count cells, and add 106 (approx 1/10 of a 25-cm2 flask) to a freshly gelatinized flask.
5. If expanding ES cells for an electroporation, where a total of 108 cells are needed, plate 3 x 106 in a 80-cm2 flask containing 30 mL of medium. Feed cells the following day with an additional 20 mL of medium. Once the cells reach confluence, trypsinize and plate 5 x 106 into three 175-cm2 flasks containing 50 mL of medium each. On the next day, add an additional 40 mL of medium. Each confluent flask should yield about 8 x 107 cells.
3.1.3. Freezing ES Cells
1. Trypsinize 25-cm2 flask as in Subheading 3.1.2., steps 2 and 3.
2. Collect trypsinized cells in 9 mL of medium and spin down at 260g for 3 min.
3. Resuspend cell pellet in 1 mL of freezing solution, and dispense 0.5 mL of suspension into two 1-mL cryotubes.
4. Freeze cells at -80°C overnight and transfer to liquid nitrogen for long-term storage.
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