Generation of Cd4-RHOA^G17Vtransgenic mice. (A) Schematic representation of the transgene construct, which includes the murine Cd4 minimal enhancer fused to the human CD4 minimal promoter followed by the murine Cd4 exon 1, which consists of 5′ UTR, intron 1, and exon 2, with the human RHOA transgene inserted 5′ to the endogenous ATG. E, murine Cd4 minimal enhancer; P, human CD4 minimal promoter; I, exon 1; II, exon 2. (B) Quantitative PCR of genomic DNA from mouse tail DNA was performed using SYBR green analysis of the RHOA transgene compared with endogenous Tet2 (P = .6953) or RhoA (P = .4444). (C) Quantitative PCR of transcripts from tgRhoA CD4⁺ lymph node cells using amplification and probe-based detection of sequences specific for mouse or human RhoA (112 253 vs 64 819; P = .0006). (D) Immunoblot analysis of whole thymus extracts from tgRhoA or control littermates. Each lane represents whole thymic extracts from a single WT or tgRhoA mouse. The RhoA antibody cross-reacts with human and mouse proteins, which have equivalent molecular weights. (E) Flow cytometric evaluation of tgRhoA or control littermate thymuses. Data are representative of 3 independent experiments. (F) Flow cytometric analysis of splenic CD4⁺ and CD8⁺ cells of tgRhoA and control littermates. Data are representative of 3 independent experiments. All P values calculated by t test with Welch’s correction.