Targeted disruption of GITR gene.

(A) Structure and restriction map of GITR gene locus (WT, top), targeting vector (VEC, middle), and predicted mutated allele (REC, bottom). The whole gene, about 600 base pairs of the promoter region and 1.2 kb of the 3′ side of GITR gene, were replaced by a neomycin resistance cassette (neo^r) in reverse orientation. Exons are represented by filled boxes. The targeting vector contains the neo^r cassette and the HSV-tk gene (TK) allowing the positive-negative selection of the ES clones. (Bi) Southern blot of ES DNA upon a BamHI digestion probed with the 3′ external probe indicated in panel A. The wild-type band is 9-kb long, while the mutated band is 11.5 kb. (Bii) The same Southern blot has been reprobed with a neomycin probe that hybridizes only to the mutated band. (C) Tail analysis of heterozygous (GITR^+/−), homozygous knock-out (GITR^−/−), and homozygous wild-type mice (GITR^+/+) by the same Southern blot as for ES cells. (D) Flow cytometric analysis of purified T cells from GITR^+/+ and GITR^−/− mice with anti-GITR antibody. The anti-GITR antibody (R&D Systems) was used undiluted (10 μL per sample), and the antigoat IgG FITC-conjugated secondary antibody was used diluted 1:100. (E) Lymphocyte subpopulations of GITR^−/− and GITR^+/+ mice. Values represent the percentage ± SD (n > 6). Cervical, brachial, axillary, superficial inguinal, and mesenteric lymph nodes were analyzed.