The method involves close collaboration with an investigator who will do all the mouse breeding to generate 4-5 plugged females by natural mating. The investigator should use the following breeding regime:
The females are brought up to the investigator’s lab and I do the dissection, flushing, embryo culture, embryo disaggregation, and culture of any resulting ES cell lines.
A rough outline of a single mating and derivation cycle is detailed. If a second round of mating is required, it is staggered by a week following the first round.
Sunday: Investigator's lab sets-up matings
Monday: Check plugs. Set mated females aside. Unmated females can be used again (0.5 d.p.c.).
Tuesday: 1.5 d.p.c.
Wednesday: 2.5 d.p.c. Plate out PMEFs for embryo culture.
Friday: day 1.
Saturday: day 2.
Sunday: day 3.
Monday: day 4.
Tuesday: day 5.
Wednesday: day 6--Primary embryo outgrowth disaggregation. (Hardest part). Disaggregations put into 4-well PMEFs with FBS-medium.
Thursday: day 7. Change medium to SR-medium.
Friday: day 8.
Saturday: day 9.
Sunday: day 10.
Monday: day 11. Critical decision day. Is there anything worth passaging? Secondary passaging day for most outgrowths, using FBS- medium onto 4-well PMEFs. Each secondary becomes an individual cell line.
Tuesday: day 12. Switch to SR-medium and feed daily thereafter. Stragglers?
Wednesday: day 13. Stragglers? Secondaries from Monday (d.11) may be ready to passage soon. Begin passaging onto 35mm PMEFs as required. Passage in FBS-medium. Next day switch to SR-medium and feed daily thereafter.
Thursday: day 14. Passaging? Stragglers?
Friday: day 15. Passaging? Stragglers?
Saturday: day 16. Passaging? Stragglers?
Sunday: day 17. Passaging? Stragglers?
Monday: day 18. Last chance for any stragglers. Passaging?
Freeze individual lines in vials as appropriate, removing aliquots for DNA. Evaluate DNA aliquots for likelihood of adaptation to growth on gelatinized plastic.
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