Two independent Capn2+/- ES cell lines, designated ES27 and ES36, were isolated from a screen of 305 drug-resistant clones. Correct targeting of the Capn2 locus was established both by Southern blot hybridization and PCR analysis. A probe located outside the short (upstream) arm of homology hybridized to a 3.5-kb BamHI fragment of the wild-type allele and 5.3-kb BamHI fragment of the mutant allele as predicted from genomic maps (Figure 2A). The same probe also detected the expected 7.2-kb wild-type and 6.4-kb mutant BglII fragments, 4.9-kb wild-type and 5.7-kb mutant NcoI fragments, as well as 7.2-kb wild-type and 4.9-kb mutant BglII/AgeI fragments (not shown). A probe derived from the PGK-Neo cassette recognized only the 5.3-kb BamHI fragment in Capn2+/- ES cells, suggesting that the targeting vector had integrated solely at the Capn2 locus (not shown). A PCR screening method was also established that generated a wild-type product of 2,749 bp and a 2,711 bp product from the mutant allele. The 2,711 bp product was only evident in the two targeted cell lines (Figure 2B).