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Fig. 1. Targeted disruption of the mouse Osr2 gene. (A) The Osr2 gene consists of four exons spanning ~8 kb of genomic DNA. Boxes indicate exons, with the protein-coding region marked in black. The positions of the translation start (ATG) and stop (TAG) codons are also indicated. Restriction sites are: B, BamHI; E, EcoRI; H, HindIII; P, PstI; X, XbaI. The targeting vector used the 2.2 kb XbaI-PstI fragment containing the intron 1/exon 2 junction as the 5' arm and the 3.3 kb XbaI-HindIII fragment 3' to the Osr2-coding region as the 3' arm. A modified bacterial lacZ gene and a neo expression cassette were inserted in between the arms and a diphtheria toxin A (DTA) expression cassette was cloned 3' to the 3' arm for negative selection against random integration. Correct targeting results in the lacZ gene and the neo cassette replacing most of the Osr2 coding region, from the sixteenth codon of the open reading frame to the XbaI site in the 3' untranslated region. Arrowheads above the wild-type and mutant genomic schematics indicate the positions of PCR primers used for genotyping. (B) Southern hybridization analysis of tail DNA samples from a litter of F1 progeny of a chimeric male generated with a targeted ES clone. Tail DNA samples were digested with BamHI, separated by electrophoresis through a 1% agarose gel, transferred onto a Zetaprobe nylon membrane (BioRad), and hybridized with random prime-labeled probes made from the 600 bp HindIII-EcoRI fragment isolated from the Osr2 genomic region 5' to the targeted region. The 14 kb BamHI fragment corresponding to the wild-type allele was detected in all F1 progeny, while the 7.7 kb mutant allele-specific fragment was detected only in heterozygotes. (C) PCR analysis of tail DNA samples from a litter of newborn F2 progeny. The fragments amplified from wild-type and mutant alleles are 490 bp and 460 bp, respectively. Homozygous mutants were born at the expected Mendelian frequency (25%). m, DNA fragment size markers; +/+, wild type; +/-, heterozygote; -/-, homozygote.





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