Homologous recombination in the murine γ-sarcoglycan locus. (a) The strategy for replacing exon 2 of murine γ-sarcoglycan with neomycin phosphotransferase (neo) is shown. The probe used for Southern blotting and the primers (arrows) used for genotyping progeny from gsg
+/− crosses are indicated. E, EcoRI; H, HindIII; N, NotI; Xb, XbaI; Xh, XhoI. (b) Southern blot of EcoRI-digested genomic DNA from ES cells. Homologous recombination in the γ-sarcoglycan locus produces the loss of a single EcoRI site. (c) PCR genotyping of progeny from gsg
+/− interbreeding. The PCR product generated by the mutant allele is 410 bp, and that generated by the wild-type allele is 376 bp. (d) Immunoblotting of total protein from wild-type (lanes 1 and 5) and mutant gsg−
/− (lanes 2 and 6) muscle with a monoclonal antibody directed at γ-sarcoglycan amino acids encoded by exon 6 (NCL-35DAG, lanes 1 and 2), or with a polyclonal anti-dystrophin antibody (AB6-10, lanes 5 and 6). No γ-sarcoglycan is detectable in gsg
−/− muscle. Dystrophin is normal in gsg
−/− muscle. Polyclonal antisera raised to the complete γ-sarcoglycan protein also shows the absence of γ-sarcoglycan protein in gsg
−/− mice (data not shown). A Coomassie-stained gel, gsg
+/+ (lane 3) and gsg
−/−(lane 4), demonstrates equivalent loading. γ-sarcoglycan and dystrophin were resolved on 12.5% and 5% SDS-PAGE, respectively.