Generation of lox-Piga-lacZ ES cells. (A) Top panel shows the genomic structure of the mouse Piga gene. The internal probes used for Southern blot analysis map to exon 1 (probe 1) and to exon 2 (probe 2), and the external probe maps to exon 6 (probe 6). Middle panel displays the targeting construct (lox-Piga-LTNL-lacZ.). Bottom panel shows thePiga gene after homologous recombination (lox-LTNL-Piga-lacZ). Shaded boxes represent exon 1-6, arrowheads represent loxP sites, hatched line represents the selectable marker cassette and the bold black line represents the lacZ gene. LTNL = loxPthymidine kinase, neomycin resistance geneloxP. Restriction enzyme sites S=SmaI, B=BamHI, E=EcoRI. (B) Southern blot of homologous recombined ES clone 1G2. Lanes 1, 3, and 5 represent DNA from wild-type ES cells and lanes 2, 4, and 6 represent DNA from recombined ES clone 1G2. Lanes 1 and 2 were digested with BamHI and hybridized with probe 6. DNA in lanes 3 and 4 was digested with EcoRI and hybridized with probe 1. DNA in lanes 5 and 6 was digested withBamHI and hybridized with probe 1. Because Piga maps to the X chromosome and the ES cells are of male origin, no other allele remains. (C) Top panel shows thelox-LTNL-Piga-lacZ gene. Bottom panel shows thelox-Piga-lacZ gene after Cre-mediated excision of the selectable marker cassette LTNL. Dashed line represents the desired Cre-mediated excision. (D) Southern blot of the desired Cre-mediated recombination. Lanes 1 and 4 represent DNA from wild-type ES cells, lanes 2 and 5 represent DNA from recombined ES clone 1G2, and lanes 3 and 6 represent DNA from Cre recombined ES clone 1G2/87. DNA in lanes 1-3 was hybridized with probe 6; DNA in lanes 4-6 was hybridized with probe 2; DNA in lanes 1-6 was digested with BamHI.