Generation of sgk1^–/– mice. (a) Targeting strategy. The neomycin resistance cassette (gray box) was flanked by two loxP sites (ovals) and inserted into intron 11. Exons 4–11, which code for the Sgk1 kinase domain (open boxes), were “floxed” by inserting a third loxP site into intron 3. N indicates NheI restriction sites, and the small black bar indicates the external 5′ probe used for Southern blot analysis. Expected fragment sizes of the wild-type and targeted sgk1 locus are also indicated. One homologously recombined ES cell clone was transiently transfected with Cre recombinase, and a clone that had undergone recombination between the first and the third loxP site (type I recombination) was chosen for injection. Arrows below the gene indicate PCR primers used for genotyping. Numbers between the arrows indicate the size of the amplified fragments. Crossed bars below a indicate homologous recombination. (b) Southern blot of NheI-digested genomic DNA from ES cell clones after gene targeting hybridized with a 5′ external probe (black bar in a). Lane 5 shows a targeted ES cell line. (c) Genotyping by PCR of genomic tail DNA of homozygous (–/–) and heterozygous (–/+) sgk1-deficient mice and wild-type mice (+/+) using a mix of three specific primers (arrows in a). (d) Autoradiograph of Northern blot analysis of Sgk1-specific transcripts in +/+ and –/– mice. The deletion of the kinase domain from the genome results in a size reduction of 0.9 kb at the mRNA level in sgk1^–/– mice.