Isolation and characterization of Capn2 targeted ES cell clones
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).
Figure 2  Characterization of targeted ES cell lines. (A) Targeted disruption of the Capn2 locus was detected initially by Southern blotting. Membranes were blotted with BamHI-digested genomic DNA extracted from ES cells and hybridized with a DIG-labeled 823 bp BamHI/HindIII fragment located immediately upstream of the short arm of the targeting vector (Figure 1). A 3.5-kb BamHI fragment corresponding to the wild-type allele was present in all cells, whereas a 5.3-kb fragment from the mutant allele was detected in two targeted cell lines, designated ES27 and ES36. (B) PCR genotyping was carried out with two separate reactions designed to amplify either a 2,748 bp segment from the wild-type allele or a 2,711 bp segment from the mutant allele. Both reactions used a common sense primer located in intron 4, outside the short arm of the targeting vector, and distinct allele-specific antisense primers. The reaction to detect the wild-type allele used an antisense primer located in exon 7 while the amplification of the mutant sequence was done with an antisense primer in the PGK-Neo cassette. The results confirm the presence of the wild-type allele in all cells, whereas the mutant allele signal was observed only in the two targeted clones. (M) denotes the molecular weight marker.