Expression of LacZ transgene in developing T cells after rAAV-mediated gene transfer. CD34⁺CD2⁻ cells were cultured on thymic stroma either with rAAV-LacZ at MOI of 10 or medium alone (control). CD2⁺ cells were FACS purified after 21 days and aliquotted for analysis of expression. (A) β-galactosidase activity: triplicate samples of 5 × 10³ cells per well were incubated with X-gal at 150 μg/mL and enumerated by phase contrast microscopy with ×40 objective magnification. (B) Detection of LacZ mRNA in bulk or CD2⁺ T cells by RT-PCR. mRNA was prepared from equivalent numbers of cells and cDNA generated by RT with random hexamers. The products were amplified by PCR with primers specific for an internal region of LacZ or Gs_α . Specificity of the PCR products was confirmed by Southern blot hybridization with a ³²P-labeled probe specific for LacZ. A Hela cell line stably transfected with LTR-LacZ was used as a positive control, and analysis was repeated for three independent experiments.