Chga gene targeting. (A) Chga wild-type gene structure. Chga from the isogenic mouse 129/SvJ strain showing the positions of wild-type Chga’s 8 coding exons, as well as restriction sites and the locations of the 3 Chga segments (arms) used to construct the targeting vector. Also shown is an undisrupted, wild-type ˜13-kbp diagnostic BamHI (asterisk) fragment, spanning the promoter, exons 13, and corresponding introns. (B) Structure of the homologously targeted Chga gene. A Southern blot probe (˜1.3 kb BamHI/XhoI fragment) detected a ˜7.3 kbp BamHI (asterisks) fragment after homologous recombination. (C) Southern blot results. While the probe (B; ˜1.3 kbp BamHI/XhoI fragment) detected only a ˜13 kbp fragment in wild-type DNA (A), it detected both ˜13-kbp (wild-type) and ˜7.3-kbp (targeted) BamHI fragments in ES cell genomic DNA after homologous recombination disrupted 1 Chga allele. Lane 1: 1-kbp extension ladder; lane 2: wild-type ES cells (13-kbp band only); lane 3: clone B7 ES cells with homologous recombination in 1 Chga allele; lane 4: clone C5 cells with homologous recombination in 1 Chga allele. (D) Chga gene after homologous integration of the targeting construct into mouse genomic DNA (Chga locus on mouse chromosome 12), followed by Cre-loxP–mediated recombination (type I deletion, between the first and third loxP sites; dashed line). The triangles represent loxP recognition sites. Asterisks indicate BamHI sites flanking the diagnostic 7.3-kbp BamHI fragment.