Figure 2.

Generation of JP-1 knockout mice. (A) Homologous recombination at the mouse JP-1 locus. Restriction enzyme maps of the wild-type allele, targeting vector, and mutant allele are illustrated. The exons in the gene (E1 and E2) are indicated by filled boxes, and the neomycin resistance gene (neo) and diphtheria toxin gene (DTA) are indicated by open boxes; the directions of transcription are shown by arrows. The hybridization probe and PCR primers for detection of the mutant gene are indicated by a hatched box and opened arrows, respectively; the predicted sizes of the DNA fragments in Southern blot analysis and PCR are also shown. (B) Detection of mutant JP-1 allele by PCR. Genomic DNAs from newborn mice were analyzed by PCR using primers indicated in A. Amplified DNA fragments were run on a 1.5% agarose gel, and size markers are indicated in base pairs. Positions of PCR products from wild-type and mutant genes are given. Immunoblot analysis of JP-1 (C) and JP-2 (D) in newborn mice. Microsomal proteins from hindlimb preparations were analyzed using specific antibody against JP-1 in C or JP-2 in D. Positions for target proteins are given, and size markers are indicated in kilodaltons. (E) Immunofluorescence detection of JP-2 in JP-1 knockout muscle. Cryosections of hindlimb muscles from wild-type and JP-1 knockout neonates were examined using an antibody specific to JP-2. No difference in staining pattern was detected between the genotypes. The partial sequence data for the JP-1 gene has been deposited in the database (GenBank/EMBL/DDBJ accession no. AB024446). Bar, 3 μm.