Fig. 5. TCRbenhancer (Eb) mutations impair Db-Jb rearrangements and thymocyte development. (A) Schematic diagrams of the murine TCRblocus (not to scale), targeting vector, and targeted allele before and after Cre/loxP-mediated deletion of the neo. Gene segments and Ebare labeled. Gal4, the inserted eight copies of Gal4 DNA sequences; open triangle, loxP site; A, ApaLI; P, PvuII, and Ps, PstI. Arrows indicate the locations and directions of primers used for PCR assays of Db-Jb rearrangements in B. (B) PCR assays for Db1-Jb 1, Db1-Jb 2, and Db2-Jb2 rearrangements. DNA was isolated from thymocytes of wild-type (+/+), Eb -^/- , and Eb ^R/R mice at 6?8 weeks of age. Semiquantitative PCR were carried out as described in Materials and Methods. DNA from wild-type thymocytes was either undiluted (undil) or serially diluted every 5-fold into recombination activating gene (RAG)2-^/- kidney DNA and then amplified to determine the linear range of the PCR assays. JAK3 was amplified to verify DNA quality and relative amount. PCR products were separated on agarose gels and hybridized with specific Db probes. Rearrangements to different Jb are labeled. G.L., germ-line. The PCR assays were performed four times on Eb -^/- DNA and six times on two independently isolated DNA from wild-type and Eb ^R/R mice. One set of representative data is shown.(C) Effect of the Eb^R/R mutation on thymocyte development. Thymocytes from »6-8-week-old wild-type and Eb^R/R mice were stained for CD4, CD8, CD44, CD25, and TCRg d. CD4 and CD8 staining profiles are shown for total live thymocytes (Left). CD44 and CD25 staining profiles are shown for DN thymocytes (Center). TCRgd expression is shown for DN and DP thymocytes (Right). Numbers outside plots indicate average total numbers of thymocytes from four to five mice. Numbers inside the plots indicate the percentages of cells in the gated areas.