Introduction of loxP sites into the Stat5a/b locus. (A) The BAC clone spans approximately 210 kb and encompasses five known genes and one new gene. The Stat5a gene is 30 kb, Stat5b is more than 54 kb, and Stat3 is more than 37 kb. The Hcrt and D11lgp1 genes are downstream of the Stat5b gene, and the Ptrf gene is upstream of the Stat3 gene. The gene sizes and intergenic sequences are drawn to scale. (B) Restriction map of the wild-type allele. B, BamHI; RI, EcoRI. (C) The Stat5b-targeting construct contains a 7.2-kb genomic sequence with a pLoxpneo cassette, and the Stat5a-targeting vector contains an 8.6-kb genomic sequence with a pLoxphyg cassette. Neomycin (neo), hygromycin (hyg), and thymidine kinase (TK) are positive and negative selection cassettes, respectively. Prior to electroporation, each targeting vector was linearized at the unique NotI site (N) indicated. X, XbaI; A, Asp718. (D) Restriction map of the targeted allele. Two loxP sites and the neomycin cassette were introduced downstream of Stat5b, and two loxP sites with the hygromycin cassette were inserted downstream of Stat5a. The indicated probes were used to assess recombination events. (E) Structure of the deleted Stat5a/b allele. D11lgp1 and Stat3 are next to each other, with one loxP site between them. (F) Southern blot analysis of homologous recombination in ES cells electroporated with the Stat5b-targeting vector. The BamHI (lanes 1, 2, and 6) restriction fragment size changes from 5.8 to 3.0 kb, as seen in the targeted cells with the 32P-labeled 500-bp external probe 1. (G) ES cell clone 1 was chosen to be electroporated with the Stat5a-targeting vector. The EcoRI (lanes 1, 3, 5, and 7) restriction fragment size changes from 15 to 2.8 kb, as seen in the targeted cells with the 32P-labeled 500-bp external probe 2. (H) Southern blot analysis of tail DNA from chimeric males (lanes 1 and 2) and their agouti pups (lanes 3 to 12). Both Stat5a- and Stat5b-targeted bands were detected in the male chimeras (lanes 1 and 2) and in four agouti pups (lanes 6, 8, 9, and 12).