Targeted disruption of the ATF4 gene.

(A) Diagram of the murine ATF4 gene, the replacement vector, and the homologous recombinant allele. The exons of the ATF4 gene are represented by solid boxes. Plasmid sequences are indicated by open boxes. A 9.9-kb BamHI-SmaI 5′ homology fragment and a 1.3-kb BamHI-SacI 3′ homology fragment were used to construct a gene-replacement targeting vector in pNTK containing the neomycin phosphotransferase cassette (neo). Broken lines indicate the regions of homology used for homologous recombination. The sites at the ends of both homology fragments were removed as they were subcloned. An EcoRV site was added at the 3′ end of the 5′ homology fragment. SalI andEcoRV sites were added to the 5′ end of the 3′ homology fragment. Locations of the 5′ and 3′ probes used for Southern blot analysis are indicated by labeled lines below the maps. The locations and sizes of the fragments produced by restriction-enzyme digestions used in Southern blot analysis are indicated below the maps: B indicates BamHI; E, EcoRV; Sa,SacI; S, SalI; Sm, SmaI; and X, XbaI. (B) Typical Southern blot analysis of progeny from heterozygote matings after EcoRV restriction-enzyme digestion and hybridization with the 3′ probe shown in panel A. Positions of diagnostic bands are indicated at left. The probe detected bands of 3.3 and 2.2 kb corresponding to the sizes predicted for the wild-type and homologous recombinant alleles, respectively. Genotypes are shown above each lane. (C) Typical Southern blot analysis of progeny from heterozygote matings afterXbaI-SalI double restriction-enzyme digestion and hybridization with the 5′ probe shown in panel A. The probe detects bands of 11.4 and 12.2 kb corresponding to the sizes predicted for the wild-type and homologous recombinant alleles, respectively.