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.