Gene targeting of the mouse Tgif locus. (A) Gene targeting strategy utilizing homologous recombination in ES cells. The wild-type allele, targeting vector, and targeted allele are shown with relevant restriction sites and exons (noncoding sequence, open boxes; coding exons, gray boxes). The dashed lines depict regions of homologous recombination. (B) Southern blot analysis of mouse MEF cells demonstrated correct targeting of the locus. EcoRI-digested genomic DNA generated a 13-kb band for the wild-type allele and a 7-kb band for the targeted allele using the 5′ probe indicated in panel A, while PstI-digested genomic DNA generated a 7-kb band for the wild-type allele and a 5.5-kb band for the targeted allele using the indicated 3′ probe. Genotypes are indicated above the lanes. (C) RT-PCR using primers indicated in panel A demonstrated that Tgif transcripts were expressed in Tgif+/+ and Tgif+/−, but not Tgif−/−, MEFs. RT is present (+) or absent (−) in each sample.