Mouse R1 ES cells [49] were maintained on gelatin-coated plates with feeder layers of mouse embryonic fibroblasts at 37°C under 5% CO2 in ES cell medium (DMEM [high glucose] supplemented with 15% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 μg/ml amphotericin, and 1,000 U/ml of ESGro [Chemicon]). Fetal bovine serum from HyClone Laboratories Inc (Logan, Utah) was tested for its ability to support ES cell growth. Gelatin was from Sigma-Aldrich Canada (Oakville, Ontario). Unless otherwise specified, all other tissue culture reagents were from Gibco-BRL.