For Southern hybridization, a 5′ external 32P-labeled probe was generated by random priming (RadPrime; Gibco BRL, Gaithersburg, MD) of a PCR fragment (oligonucleotides Ex5(+)/Ex6(—); 538 base pairs [bp] in length) of nectin-2 cDNA. Eight ES clones that were positive by both screening procedures were injected into C57BL/6 blastocysts to generate chimera. One clone, EW63, resulted in highly chimeric male offspring, one of which transmitted the targeted nectin-2 locus through the germline when mated to DBA2XC57BL/6 hybrid females. Male and female offspring carrying the targeted allele were intermated to produce homozygous animals.
After the correct targeting of the founder line was established by the procedure outlined above, routine genotyping of nectin-2 mice was done by a simpler genomic PCR analysis using a mix of three oligonucleotides, MPH126(+)/ LacZ135(—)/Ex2-3′(—). Cycling conditions were 30 sec at 96°C, 45 sec at 60°C, and 45 sec at 72°C for 28 cycles. Whereas the 210-bp PCR reaction product of MPH126(+)/LacZ135 identifies the targeted allele, the 350-bp PCR product MPH126(+)/Ex2-3′(—) is indicative of the wild-type (wt) allele (Fig. 1D).