Impaired T-cell development in the absence of Rac1 and Rac2. (A) Gel electrophoretic analysis showing PCR products diagnostic for the Rac1^flox (flox) and Rac1⁻ (KO) alleles amplified from DNA extracted from the indicated sorted thymocyte subsets (DN1, DN2/3, DN4, and DP) from 2 individual Rac1^TRac2^−/− mice (lanes 1,2) and, as a control for efficient amplification, from total thymocytes from a Rac1^flox/−Rac2^−/− (control) mouse that did not carry the hCD2-iCre transgene. (B) Flow cytometric analysis of CD4 and CD8 expression on thymocytes from Rac1^+/+Rac2^+/+hCD2-iCre (WT) and Rac1^flox/floxRac2^−/−hCD2-iCre (Rac1^TRac2^−/−) mice. Gates identify CD4⁻CD8⁻ double-negative (DN), CD4⁺CD8⁺ double-positive (DP), and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive (CD4SP and CD8SP) cells. Numbers indicate percentage of cells falling into the gates. (C) Flow cytometric analysis of CD44 and CD25 expression on thymocytes from WT and Rac1^TRac2^−/− mice, which were Thy1⁺ but negative for expression of the lineage (Lin) markers (CD4, CD8, CD3ϵ, TCRβ, TCRγδ, B220, Gr1, Mac1, Dx5; not shown). Gates show DN1 (CD44⁺CD25⁻), DN2 and DN3 (DN2/3, CD44^+/−CD25⁺), and DN4 (CD44⁻CD25⁻) populations. Numbers indicate percentage of cells falling into the gates. (D) Graphs showing mean (± SEM) total thymocyte cell numbers or numbers of cells in populations gated as in panels B and C. Also shown are the number of thymocytes expressing TCRγδ (γδ). Thymocytes were analyzed from WT, Rac1^flox/floxRac2^+/+hCD2-iCre (Rac1^T), Rac1^+/+Rac2^−/−hCD2-iCre (Rac2^−/−), and Rac1^TRac2^−/− mice. (E) Graph showing mean (± SEM) CD4⁺ or CD8⁺ T-cell numbers in the lymph nodes and spleen of mice of the indicated genotypes. (F) Gel electrophoretic analysis showing PCR products diagnostic for the Rac1^flox (flox) and Rac1⁻ (KO) alleles amplified from DNA extracted from CD4⁺ or CD8⁺ T cells isolated from the lymph nodes of Rac1^TRac2^−/− mice, or from tail DNA of a control Rac1^flox/−Rac2^−/− mouse that did not carry the hCD2-iCre transgene. MM indicates molecular weight markers. Statistically significant differences between WT and Rac1^TRac2^−/− mice are indicated (*P < .01; **P < .001).