Genotyping was also carried out by PCR analysis of genomic DNA. The sequences of all oligonucleotide primers are listed in Table 1. A single-step PCR strategy was sufficient for genotyping ES cells or biopsies from post-implantation embryos and weanlings (Figure 1). A 2,748 bp segment of the wild-type allele and a 2,711 bp segment of the mutant allele were amplified in separate reactions using a common (intron 4) sense primer, located outside the short arm of homology, and distinct antisense primers which hybridized to either wild-type (exon 7) or mutant (PGK-Neo) sequence (Table I). The thermocycling parameters included a five minute initial denaturation step at 95°C, 30 cycles of one minute denaturation at 95°C, one minute annealing at 56°C, and one minute extension at 72°C, with a ten minute final extension step.