Embryos used for RT PCR analysis were dissected in DMEM, 25 mM HEPES pH 7.4, 15% FCS, 2 mM glutamine, penicillin and streptomycin. Embryonic and extra embryonic tissue was removed from the ectoplacental cone and frozen in dry ice. The ectoplacental cone was grown ex vivo in DMEM, 15% FCS, glutamine, penicillin and streptomycin using standard culture conditions. After 4 days growth extra embryonic cells were removed from the outgrowth and subjected to PCR genotyping as described above and qPCR as described below.
