Figure 1.
Targeted disruption of the CCR4 gene. (a) Targeting strategy. Wild-type CCR4 locus with partial restriction map (top), targeting vector (middle), and predicted structure of the targeted allele after homologous recombination (bottom). The coding region of the gene is shown as a black box. The neomycin resistance gene is light gray and thymidine kinase gene is dark gray. The arrows denote the position of the PCR primers used to identify ES cell clones expressing the transgene. The probe used for screening genomic DNA is shown by the thick black bar (probe). Restriction sites are as follows: P, PstI; N, NheI; Ns, NsiI; A, AvrI; X, XhoI; E5, EcoRV; Ec, Eco47-3; H, HpaI; and H3, HindIII. (b) Representative Southern blot analysis of PstI-digested tail DNA from wild-type (CCR4+/+), heterozygous (CCR4+/−), and homozygous knockout mice (CCR4−/−). The expected band sizes of the wild-type allele (4.5 kb) and the targeted allele (3.4 kb) are indicated by arrows. (c) RT-PCR analysis of CCR4 mRNA. Spleen (lanes 1–4) and thymus (lanes 5–8) of CCR4+/+ (lanes 1, 2, 5, and 6) and CCR4−/− mice (lanes 3, 4, 7, and 8). (d) RT-PCR analysis of CCR4 mRNA in peritoneal macrophages isolated from CCR4+/+ (lanes 1–3) and CCR4−/− mice (lanes 4–6). The predicted band size of CCR4 PCR product indicated by the arrow is 1.1 kb. (e) Chemotaxis of splenocytes in response to CCR4 ligands. CCR4+/+ (open circles) and CCR4−/− mice (filled circles). Results shown are the means of triplicate determinations for each concentration of chemokine, and are representative of at least four experiments.