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.