Fig. 1. Generation of the Myog^flox allele, targeted ES cells and heterozygous mice. (A) The top line shows the myogenin locus with its three exons (black rectangles). The second line shows the targeting construct. The region of homologous DNA (thicker line) spans 3.8 kb and is flanked by EcoRI sites. Also shown are loxP sites (red triangles) and a neomycin cassette placed into the BamHI site in the first intron (gray rectangle). A thymidine kinase (TK) cassette (white rectangle) lies upstream of the homology region. The next two lines depict the targeted Myog locus before and after Cre recombinase-mediated deletion of the first exon and neomycin cassette. (B) Southern genome hybridization of ES cell DNA digested with BamHI and HindIII, confirming proper targeting. A 3' probe derived from the third exon immediately 3' to the EcoRI site outside of the homology region was used in the hybridization analysis. The 2 kb band shown on the figure represents the wild-type BamHI fragment bounding the second and third exons (Myog⁺). The 6 kb band represents the targeted BamHI-HindIII fragment (Myog^flox) containing the neomycin cassette. The left and right lanes show DNA from targeted and wild-type ES cell lines, respectively. (C) Southern genome hybridization of tail DNA digested with EcoRI and hybridized with a Myog cDNA probe. Tail DNA was obtained from P10 pups resulting from mating Myog^+/- and Myog^+/flox mice. The left, idle and right lanes show DNA from Myog^+/flox, Myog^+/- and Myog^flox/- mice, respectively. The 2 kb band representing the Myog^- allele is the result of a EcoRI site in the neomycin cassette that is not present in the neomycin cassette of the Myog^flox allele. The origins of the 4 kb and 6 kb bands associated with the wild-type and Myog^flox alleles, respectively, are shown in A.