Clonal expansion of blastic thymocytes with marked accumulation of Rap1GTP in the recipients of C3G-F/SPA-1^−/− HPCs. (A) Cells from the thymi of Vect/SPA-1^−/− and C3G-F/SPA-1^−/− HPC recipients were Giemsa-stained 12 weeks after transplantation. They were also multicolor analyzed for the expression of indicated antigens with the use of FACScan; the representative profiles in GFP⁺ fraction are shown. Filled areas in histograms indicate the control staining. (B) Spleen and BM cells (BMCs) of Vect/SPA−1^−/− and C3G-F/SPA-1^−/− HPC recipients were 3-color analyzed with the use of FACScan; CD4/CD8 expression profiles in GFP⁺ fractions are shown. (C) DNAs from the thymic cells of Vect/B6, C3G-F/SPA-1^−/−, and C3G-F/B6 HPC recipients 12 weeks after transplantation were digested with indicated restriction enzymes and Southern blotted with the use of an EGFP (left panel) or a TCR Cβ (middle panels) cDNA probe. Normal liver and thymocytes served as controls for negative and positive Cβ gene rearrangement, respectively. Thymocytes from a C3G-F/SPA-1^−/− HPC recipient were treated with colcemid and subjected to spectral karyotyping analysis (right panel). Eight of 10 mitotic cells showed an identical karyotype with chromosome 15 trisomy (boxed). (D) Cells from the thymi of Vect/B6, C3G-F/B6, and C3G-F/SPA-1^−/− HPC recipients 12 weeks after transplantation were lysed; Rap1 GTP was detected by a pull-down assay. Vertical lines have been inserted to indicate a repositioned gel lane.