Making embryonic fibroblasts (for for ES culture)

To make G418r EF (i.e. EFneo), mate a homozygous fertile neo containing male (e.g. beta-2m -/-) with a wild-type female; we use Balb/c. Get 2 pregnant females: it's worth processing 10-15 embryos at a time to save on work.

1) Kill pregnant female at day 13 or 14 of gestation.

2) Do dissections in sterile conditions in t.c. hood. Wash mouse with 70% ethanol. Dissect out embryos free of extraembryonic membranes. Dip in PBS. Dissect away soft organs and viscera (e.g. liver, brain, gut, etc). Remaining carcass throw into trypsin (regular ES cell 0.25% trypsin with EDTA) in a 6cm dish. Mince embryos very finely.

3)Transfer all embryos to a 50 ml tube and make up to 2ml/embryo with more trypsin solution. Put at 370C for about 30'. Pipette up and down every 5-10'.

4) Add equal volume of media (DME + 10% Foetal claf serum + Pen/Strep + Glutamine). Mix. Let big bits settie for 1-2', remove supematant and plate out.

5) Put 1 embryo/T175 flask in 50ml media. Incubate at 370C, 5% CO2

6) Change medium next day.

7) Grow to extreme confluency.

8) Freeze all flasks at this stage, except one. Freeze in 2 vials/flask.

9) Split remaining flask 5x1/5 into T175 flasks.

10) Grow to extreme confluency. Split again 5x1/5 into T175 flasks - total of 25 flasks at this point

11) Grow to extreme confluency and now freeze each flask in 5 vials after gamma-irradiation
(3500 rads - 2.5' in centre of the NIMR Co source). There will be a total of 125 vials
of irradiated EF's/embryo.

12) Should have about 4x106 cells/vial. This is enough for lxl0cm plate or 2x6cm plates.

Thus each embryo gives enough for about 2 large electroporation experiments.