Figure 1.

Generation of a Faim2^−/− mouse line by targeted gene disruption via Cre-mediated recombination in vivo. A, Targeting strategy. First, Faim2 wild-type allele a₁ displaying a 9 kb region of the minus strand of chromosome 15 containing the region of interest with exons 3–5 (white boxes), two HindIII restriction sites (H), an external 5′ 0.5 kb Southern hybridization probe (gray bar) with a predicted 5.1 kb a₁-specific fragment (gray dashed line), and the annealing loci of PCR primers 1 and 2 (gray arrows) with a predicted a₁-specific 300 bp amplicon (gray dashed line). Second, Targeting construct displaying a 5′ 0.5 kb arm of homology, an antisense-directed and directly repeated frt site (black arrowheads)-flanked neomycin-resistance gene (Neo box) including an exogenous HindIII restriction site (H), a 1.5 kb genomic region containing exons 3–5 flanked by directly repeated loxP sites (white arrowheads), and a 3′ 4.5 kb arm of homology. Third, Targeted Faim2 allele a₂ after homologous recombination displaying the 5′ Southern probe with its predicted a₂-specific 2.5 kb fragment (gray dashed line) and the annealing loci of PCR primers 3 and 4 (gray arrows), which amplify an a₂-specific 746 bp product (gray dotted line). Fourth, Disrupted Faim2 allele a₃ after Cre-mediated recombination in vivo displaying the remaining frt-flanked Neo-cassette and a single loxP site as well as the annealing loci of PCR primers 5 and 6 with their predicted a₃-specific 202 bp amplicon (gray dotted line). B, Southern blot (top) (HindIII restriction enzyme digest, 5′ external probe) and PCR (bottom) (primers 3 and 4) on tail DNA verifying homologous recombination on the a₂ allele. C, PCR [primers 1 and 2 (bottom) and 5 and 6 (top)] on tail DNA identifying a₃-mutant and a₁-wild-type allele, respectively. D, RT-PCR on brain homogenates verifying the absence of Faim2-specific transcripts in a₃-null mutants; amplicons span from exon 1 to 12, 1 to 2, 2 to 6, and 7 to 10; GAPDH was used as loading control. E, F, Northern blot (E) and Western blot (F) prepared from brain homogenates documenting the absence of Faim2-specific mRNA as well as of the full-length protein in a homozygous a₃-disrupted Faim2 genotype compared with homozygous a₁-wild-type mice and thereby verifying an in vivo Faim2 null mutant.