Outgrowth assays were preformed on E3.5 blastocysts. E3.5 blastocysts were isolated in 15% FCS, M2, 10 µM mercapto-ethanol, penicillin and streptomycin using standard procedures. Blastocysts were then transferred to gelatinized plates containing 15% FCS, M16, 10 µM β-mercaptoethanol, 2 mM glutamine with penicillin and streptomycin. Outgrowths were cultured under standard conditions for 5 days and observed for defects. At the end of the experiment the outgrowths were removed and genotyped by PCR using conditions above.
