Generation of exon3 knockout by targeted insertion of a transcriptional STOP cassette in the Fgf2 locus. A, Structure of the targeting vector, the wild type Fgf2 gene and the Fgf2 locus after homologous recombination. Boxes: exons; lines: introns; red triangles: LoxP sites; black bars: location of Southern blot probes; double-arrowed lines: size of the corresponding fragments; orange triangles: location of genotyping primers for the wild type allele; green triangles: genotyping primers for the knockout allele; blue triangles: location of RT-PCR primers to assess the expression of the βgeo product. Abbreviations: SA, Splice acceptor from Adenovirus; βgeo, β-galactosidase -Neomycin gene fusion construct; BPA, polyadenylation site; PGK DTA, Phosphoglycerate kinase promoter-driven diphtheria toxin gene. B, Southern blot images of DNA digests from the correctly targeted ES cells. Black arrows point to the targeted allele, and white arrow to the wild type allele. C, RT-PCR with primers specific for the wild type allele (upstream primer in exon 2, downstream primer in exon 3). D, RT-PCR with primers specific for the knockout allele (located in the βgal construct, blue triangles in A). E, Western blot showing absent expression of the FGF2 protein in exon3 KO mice. Wt, wild type mice; ht, heterozygous mice; ko, mice homozygous for the exon 3 truncated allele.