Ron Lab


 

ES cell culture


Medium Amount
dMEM (Gibco) 500ml
ES cell qualified serum (e.g. Gemini Cat #100-125) see www.gembio.com 90ml
Pen Strep (Gibco) 6ml
MEM sodium pyruvate (Gibco cat #11360070) 6ml
MEM Non-essential amino acids (Gibco cat #11140050) 6ml
l-glutamine (Gibco cat #25030081) 6ml
mercaptothanol (55 mMstock in D-PBS)
(Gibco cat #21985023)
600μl
Lif (leukemia inhibitory factor)

We make our own lif from a cell line that over-expresses the protein. It is available to purchase from BD sciences, Chemicon, Cedar lane, and possibly other vendors.
 

Gelatin coating of dishes:
  • Make 0.1% gelatin in dH20 and autoclave. Store at room temp.
  • Cover bottom of tissue culture dishes with solution and incubate 5-15min. Remove gelatin and plates are ready to use. Add medium before adding cells to prevent hypotonic lysis.
Feeders
  • Grow early pass (p1-p5) primary cultures of mouse embryonic fibroblasts on plates coated with 0.1% gelatin.
  • Treat confluent dishes for 2 hours with 10μg/ml mitomycin C (Sigma). The mitomycin can be saved and reused 2X within a 30 day period. Keep solution to reuse wrapped in foil at 4C.
  • After treatment, wash the cells twice with PBS and split into 2.5-3 new dishes. The treated feeders can be kept for ~1 week prior to use.
Growth of ES cells
  • ES Cells should preferentially be maintained on feeder layers in order to prevent differentiation.
  • ES Cells can be grown on gelatin coated plates for experiments.
  • Since feeders attached to uncoated plastic more quickly than ES cells, ES cell cultures can be depleted of feeders by trypsonization and replating for 10minutes on an untreated dish. After 10 minutes, the dish is tilted and non-adherent cells are transferred to a new gelatin coated dish.
  • After thawing cells will take 1-3 days to recover. The medium should be replaced every day. The cells should not be allowed to reach more than 80% confluence. Once the cultures have recovered from thawing, they can be passed 1:4 to 1:5 every day or 1:10 every 2 days or 1:20-1:40 every 3 days. The medium should be replaced as soon as it begins to turn yellow.
  • The cells should be trypsonized with ~3ml of trypson/10cm dish at 37C for ~5-8min and then neutralized with 8ml of growth medium.
Selection of homozygous clones by selection with
high concentrations of G418

Note: Homozygous mutant (knockout) W4 ES cells originally targeted at one locus with a PGK-neo based targeting vector were successfully selected with this method. Other ES cell lines or other Neo targeting cassettes may require different concentrations of G418 than indicated below. In general, the heterozygous clones were originally selected in 250-300μg/ml of G418 and double knockouts were obtained by selecting in ~5 to 10 times as much G418 as was used in the original selection.

  • Make 500mg/ml stock of G418 in 10mM HEPES pH 7.9.
  • Use rapidly growing heterozygous targeted ES cells that have been passed twice 1/5 in the original concentration of G418 (250μg/ml).
  • Plate 10cm dishes with the clone in the evening as indicated in the chart below. Two doses of cells are used to ensure that there are plates that do not become overcrowded.
  • Change the medium in the next morning (~14 hrs later) and add the indicated concentration of G418.
  • (The medium will be more yellowish than usual, but adjusting the pH does not seem to be necessary as long as the G418 was made in HEPES. Do not make in more that 10mM HEPES as this can inactivate the G418.)
  • Change the medium on the high density plates every day. As the cells die, the medium can be changed as little as every three days.
  • Colonies will generally grow slower than with normal selection. ~9-12 days until 2mm colonies are formed. Expect ~0-20 colonies on the plates with the highest doses of G418.
  • Pick 25-400 colonies (see expected results below) from plates with the highest dose of G418 to have colonies.
  • Expand colonies and make duplicate 96 well plates for freezing and DNA prep.
  • Do a primary screen for loss of both WT alleles by PCR on about 25 clones. If positives are obtained go on to the next step. If not, screen an additional 100-400 clones.
  • Confirm positives by southern because some positives may have partial duplications or other undesirable rearrangements of the targeted allele(s).
Number of dishes Number of cells G418 (mg/ml)
2 1x106 1.5
2 1x106 1.75
3 1x106 2.0
3 1x106 2.25
2 2x105 1.5
2 2x105 1.75
3 2x105 2.0
3 2x105 2.25

Expected results

In three of four of our targeted genes we obtained "clean" double knockouts at a frequency of ~10-25% of clones picked from the highest dose with surviving colonies (usually 1.75-2.25mg/ml G418). In the fourth targeted gene we had a double targeting frequency of only ~1.3%.

Heather P. Harding
NYU School of Medicine
April 9, 2003