EMBRYONIC STEM CELL CULTURE-GENE TARGETING

Things to do before thawing the ES cells

1. Make your KO construct. We generally use pPNT or one of its derivatives (pLNT with loxP sites, pPNTFRT with FRT sites around the neo cassette etc.) Make absolutely sure that it is correct! Also, make sure that you have a good probe for a Southern blot (mandatory!) and a good strategy for PCR genotyping if you want.

2. Make mitomycin C inactivated MEFs. Obtain regular MEF cells and expand them (how far you expand them will depend on the source!). To inactivate MEFs, add 100 ml mitomycin C (10 mgs/ml) to each 10 cm plate of MEFs containing 10 mls of MEF media for 2 hours. After 2 hours, remove the media, trypsinize the cells and freeze into 1 vial per 10 cm plate. When you thaw, thaw each vial into a 10 cm plate to be used for ES cells.

3. Remember that ES cells require attention EVERY day until they are frozen. Make sure that you plan accordingly-don’t start them right before the holidays if you are not going to be here every day! (even Christmas!).

This protocol is based on R1 and RW4 cells. Other ES cell lines may not behave the same way!

1. Thaw ES cells into a 10 cm plate with MEFs and 12 mls of ES media.

2. The next day change the media.

3. After 2-3 days, split ES cells into 4 to 6 10 cm plates with MEFs. Will need 2X10⁷ cells per electroporation-the rest you can freeze.

4. On day of electroporation, trypsinize cells, spin down and wash once with ES growth media and once with DMEM without serum or additives.

5. Resuspend 2X10⁷ cells into 0.8 mls final volume of DMEM without serum or additives. Add 40 mgs of linearized KO construct and mix. Transfer to a 0.4 mm cuvette.

6. Electroporate under the following conditions: Bio-Rad Gene Pulser-

250 volts, 500mF.

7. Let cells sit for 2-3 minutes at room temperature and then remove them from the cuvette gently and add to 9 mls of ES growth media.

8. Add 2 mls of electroporated ES cells to each of 5 10 cm plates with MEFs (containing 10 mls of ES growth media). Let cells recover: for R1 cells, incubate 48 hours (changing the media after 24 hours) and for RW4 cells incubate 24 hours.

9. After the recovery period, add ES growth media containing 250 mgs G418 and 1X FIAU or 1 mM gangcyclovir. Select cells for a minimum of 7 days and a maximum of 10 days changing the selection media every day.

10. Using a pipetman, pick clones into a 96 well plate containing 50-100 ml trypsin. After a few minutes (NOT TOO LONG!) transfer the picked clones into a 24 well plate containing MEFs and 2 mls of ES growth media. Incubate until you see the cells grow up (5-7 days) changing media every day (1 ml).

11. As different clones grow, split them 50/50 into 1-24 well plate with MEFs and 1- 24 well plate that has been coated with a 0.1% gelatin solution for 15 minutes at RT. After 2 days, freeze the 24 well plates with the ES cells on MEFs by removing the growth media and adding 0.5 ml of freezing media (70% ES growth media, 20 % serum, 10% DMSO), wrapping the edges with Parafilm and freezing at —80 in a Styrofoam box. After 24 hours, put the ES cells/MEFs into the —140 freezer. The ES cellson the gelatin coated plates will be processed for genomic DNA production (see associated protocol). Screen clones-Good Luck!

MEF media

450 mls of DMEM, high glucose, w/glutamine

50 mls of Hyclone serum

5 mls of penn/strep

5 mls non-essential amino acids

5 mls 100X glutamine

R1 ES media

400 mls DMEM, high glucose, w/glutamine (Gibco)

75 mls Hyclone serum

5 mls pen/strep

5 mls non-essential amino acids

5 mls sodium pyruvate

5 mls 100X glutamine

4 ml b-mercaptoethanol

50 mls ESGRO (LIF)