Isolation of pre-implantation embryos
Time of fertilization was determined by observation of copulation plugs, and noon of that day was defined as E0.5. Pre-implantation embryos were obtained by dissecting the uteri out of pregnant females at E2.5 or E3.5 and flushing the oviducts with ES cell medium. Isolated embryos were then digested for five hours at 55°C in 20 μL of lysis buffer (50 mM Tris-HCl, 0.5% Triton X-100, 200 μL/mL proteinase K, pH 8.0), followed by ten minutes at 95°C to inactivate the proteinase K. Lysates were then used for PCR genotyping.