FIG. 1.
Targeted disruption of the mouse Bard1 gene. (A) A partial restriction map of the genomic region encompassing Bard1 exon 1 (Ex1; black box) is shown on top, followed by a diagram of the targeting vector used for insertion of the hygromycin resistance-EGFP gene cassette (HygEGFP) into exon 1 of the mouse Bard1 gene (bottom). A diphtheria toxin A gene (DT) was also included in the targeting construct for negative selection. The wavy lines represent plasmid sequences. Relevant restriction sites are EcoRI (E), HindIII (H), PstI (P), and SalI (S). The 5′ flanking probe used for Southern analyses, the sizes of the endogenous and expected DNA fragments, and the locations of the PCR primers a, b, and c are also shown. hyg, hygromycin resistance gene. (B) Schematics of the neomycin resistance gene (neo) targeting vector and the recombined Bard1 locus are shown. (C) Southern analysis of ES cell DNA digested with PstI to confirm gene targeting. Due to an additional PstI site in the hygromycin selection marker, the 7.2-kb PstI fragment detected in wild-type ES cells is reduced to 5.5 kb in properly targeted ES cells. (D) Southern analysis to identify the second targeting event in heterozygous ES cells. wt, wild type. (E) Representative PCR genotyping of E6.5 embryos with primers a, b, and c. PCR amplification was conducted on embryonic DNA from wild-type (lanes 1 and 2), Bard1+/− (lane 3), and Bard1−/− (lane 4) embryos. The PCR products derived from wild-type and mutant alleles are 347 and 422 bp, respectively.