Generation and identification of CCR9-deficient mice.

(A) Partial restriction map of the wild-type CCR9 gene (i). ░, exons 2 and 3. The restriction sites are RI, EcoRI, and H,HindIII. Targeting vector used for the deletion of exon 3 (ii). Two diagnostic HindIII and EcoRI restriction sites were introduced at each end of the loxP-flanked neomycin resistance gene (neo). Open boxes correspond to the thymidine kinase expression cassette (tk), to the loxP-flanked neomycin gene, and to the pBluescript IIKS+ vector (pBS). Structure of the targeted allele following homologous recombination (iii). Final structure of the targeted allele after removal of the neomycin resistance gene via cre-mediated recombination (iv). The 5′ and 3′ single-copy probes used to verify the targeting events are indicated as ▪, and the position of the primers used to monitor the germline transmission of the intented mutation is indicated by arrows. (B) Southern blot analysis of the recombinant ES cell clone VC9 that gave germline transmission prior to deletion of the neomycin resistance gene. DNA was digested withEcoRI and hybridized with the 3′ single-copy probe. (C) DNA-PCR analysis of wild-type (+/+), neomycin-deleted heterozygous (+/−), and neomycin-nondeleted heterozygous (+/neo) littermates using the pair of primers shown in (A). After deletion of the neomycin resistance gene, the targeted CCR9 allele gave an amplified PCR product of 330 bp. Amplified products were run on an agarose gel and stained with ethidium bromide.