FIG. 1.
Generation and confirmation of MyocY423H mice. (A) We generated a construct carrying the targeted mutation MyocTyr423His and selected for homologous recombination by growing cells in the presence of G418/FIAU and neomycin. Cells with a correctly targeted Myoc gene were confirmed by Southern blotting. Correctly targeted cells have a restriction fragment that is 2 kb larger than nontargeted cells when tested with both a 5′ probe and a 3′ probe indicating the presence of the neo cassette. Following production of heterozygous targeted mice, the neo cassette was removed by crossing the carrier mice to CMV-Cre mice carrying Cre recombinase under control of the CMV promoter. WT, wild type. (B) Direct sequence analysis of cDNA isolated from tissue enriched for iridocorneal angle confirmed the presence of a transcript from the targeted MyocY423H allele in both heterozygous and homozygous mutant mice. (C) Western analysis with a MYOC-specific antibody confirmed that the expressed mutant allele is translated and that mutant MYOC is present. The presence of smaller bands in the MyocY423H/+ and MyocY423H/Y423H mice indicates that the mutant protein has properties that differ from those of normal MYOC. As expected, no band was detected in mice homozygous for a null mutation (Myoc−/−) (23).