Fig. 1

Strategy for targeted mutation of the RelA gene. a The open boxes represent exons 1–4, and 9–11; the solid boxes represent exons 5–8. The solid lines represent intronic sequences; the dashed line represents plasmid vector DNA. The triangles represent loxP sequences and the ovals represent frt sequences. The gene-targeting vector possesses a total of 7.2-kb homologous sequence. The PGKneobpA cassette was inserted into an EcoRV site located ~0.6-kb upstream of exon 5. The second (stand-alone) loxP was inserted into an AflII site located ~240-bp downstream of exon 8. The vector has a 2.9-kb 5′-homology arm, and a 3′-homology arm divided into 2.7-kb and 1.7-kb segments. Homologous recombination between WT RelA and the targeting vector may result in a RelA mutant allele that carries both PGKneobpA cassette and the stand-alone loxP [Targeted (floxed-neo) allele], or one that carries only PGKneobpA cassette [Targeted (neo) allele]. The 5′-flanking probe hybridized to EcoRV-digested genomic DNA detects 4.8-kb WT bands, and 6.7-kb targeted allele bands; the 3′-flanking probe hybridized to Asp718-digested genomic DNA detects 4.9-kb WT bands, 4.1-kb targeted (floxed-neo) allele bands, and 4.9-kb targeted (neo) allele bands. b Southern blot analysis of DNA isolated from ES cell clones. Three clones, 1–60, 1–95, and 2–32, were found correctly targeted in the initial screening, and further expanded; DNA was isolated and subjected to Southern blot analysis. DNA isolated from a random integration clone was used as a non-recombinant control (Random). A restriction enzyme and a probe used in the analysis were indicated below each panel