Expression of SPA-1 transgene impairs the development of αβ T-lineage cells specifically at the DN3 to DN4 stage transition. (A) Schematic representation of the Tg vector (top). Primer positions to detect Cre-mediated recombination are indicated. MEFs from SPA-1^flox Tg mice were infected with empty or Cre-containing adenovirus and then cultured for 2 days. DNA was extracted for the genomic PCR with the use of above primers (bottom left). Aliquots of the cells were lysed and subjected to immunoblotting with the indicated antibodies (bottom right). Rap1GTP was assessed by pull-down assay. (B) Two independent SPA-1^flox Tg mouse lines, B4 and C2, were crossed with lck-Cre Tg mice, and the thymi of double Tg (lck/SPA-1) and littermate control mice were examined at 8 to 12 weeks of age. Representative macroscopic images and the mean thymocyte numbers of 5 mice are shown. *P < .01. (C) Representative FACS profiles of the thymocytes from lck/SPA-1 Tg and control littermates (top). The mean cell numbers and SE of the thymocyte subsets from 5 lck/SPA-1 Tg (■) and 5 littermate SPA-1^flox Tg (□) mice are indicated. *P < .01. Thymocytes of each subset were sorted, and genomic PCR was done. ▷ indicates a flox band and ▶ a Cre-recombined band. (D) SPA-1^flox Tg mice (C2 Tg mouse line) were crossed with CD4-Cre Tg mice, and the thymi of double Tg (CD4/SPA-1) and littermate control mice were examined at 5 to 6 weeks of age. Representative FACS profiles (left) and the mean cell numbers and SE of the thymocyte subsets from 4 CD4/SPA-1 Tg (■) and 4 littermate SPA-1^flox Tg (□) mice are indicated (right). Thymocytes of DN and DP/SP fractions were sorted, and genomic PCR was done.