Construction of the Capn2 targeting vector
A targeting construct was designed to replace a 785 bp BamHI-HindIII fragment, containing exon 7 of the Capn2 gene, with the PGK-Neo cassette (Figure 1). Exon 7 encodes 24 amino acids in the active-site region, including Asn286, one of the catalytic triad residues. The short (upstream) arm of the targeting construct was provided by a 2.7-kb HindIII-BamHI fragment, containing exons 5 and 6, which was inserted into the pPNT vector upstream of the PGK-Neo cassette [48]. During cloning, the BamHI site at the 3' end of the short arm was abolished. The loss of this BamHI site in the mutant allele, coupled with the introduction of a new BamHI site at the 3' end of the PGK-Neo cassette, provided a basis for distinguishing the wild-type and mutant alleles by Southern blotting. The long (downstream) arm of homology was provided by a 7-kb HindIII-KpnI fragment, extending from intron 7 to intron 12, inserted between the PGK-Neo and thymidine kinase (tk) cassettes.
Figure 1  Targeting strategy for disruption of the murine Capn2 gene. The murine Capn2 gene, encoding the m-80 k subunit, was disrupted in ES cells by homologous recombination. The structures of the wild-type allele (top), the targeting vector (middle), and the mutant allele (bottom) are depicted. In the mutant allele, a PGK-Neo cassette replaces a 0.8-kb genomic fragment containing exon 7 (grey rectangle) which encodes the active site asparagine residue (Asn286) In the targeting vector, the PGK-Neo cassette is flanked by 2.7-kb of Capn2 homologous sequence in the upstream (short) arm and 7.9-kb of homology in the downstream (long) arm. A probe located immediately outside of the short arm detects a 3.5-kb BamHI fragment from the wild-type allele and a 5.3-kb BamHI fragment from the mutant allele. Exons are depicted as open vertical rectangles except for exon 7 which is represented by a solid vertical rectangle. The probe used in most Southern blot analyses is shown as a solid, horizontal rectangle, while triangles mark the positions of PCR primers also used for genotyping purposes.