Fig 1.

Targeted disruption of the promoter region and of exon 1 of the Odf1 gene. (A) Structure of the wild-type allele, targeting vector, and recombinant allele are shown, together with relevant restriction sites, primer positions, and external hybridization probe position. The targeting vector consists of 5′ upstream region (∼3 kb) and intronic sequences (∼3.8 kb) flanking the pgk-neomycin selection cassette (Neo). TK, thymidine kinase cassette; X, XbaI; H, HindIII; P, PstI; E1, exon 1; E2, exon 2; I, intron; Odf1-5′ko, Odf1-I, Odf1-N, and Neomy, primers used for genotyping. Southern blot hybridization of HindIII-digested genomic DNA with the 3′ external probe resulted in an ∼5.4-kb fragment of the wild-type allele and a ∼7.2-kb fragment of the recombined allele. Genotyping with primer pair Odf1-N/Odf1-I resulted in an ∼800-bp fragment of the wild-type allele, and genotyping with the primer pair Neomy/Odf1-I resulted in an ∼700-bp fragment of the recombined allele. (B) Southern blot hybridization of HindIII-digested genomic DNA isolated from individual ES cell colonies generated by electroporation with the targeting construct, followed by selection with G418 and ganciclovir. Hybridization with the 3′ external probe to a fragment of ∼7.2 kb revealed homologous recombination. The fragment of the wild-type allele is shown at ∼5.4 kb. +/+, no homologous recombination; +/−, homologous recombination. (C) Genotyping using the primer pair Neomy/Odf1-I for detection of the recombined allele (Neo) and the primer pair Odf1-N/Odf1-I for detection of the wild-type allele (Odf1) in homozygous (−/−) and heterozygous (+/−) mice.