Lentivirus construct is present and active in mature cells derived from transduced human CD34⁺ precursors. (A) T cells were isolated from spleens and PB of mice reconstituted with transduced human CD34⁺ cells and were expanded in vitro and sorted based on the expression of GFP. Cells were then γ-irradiated. Six hours later, whole-cell extracts were prepared, separated on 10% SDS-PAGE, and immunoblotted to detect human p53. A Western blot with antibody against β-actin was used as a control. Results from 2 mice are shown. (B) T cells expressing the p53i or control-GFP constructs were treated with different stimuli and assayed 24 hours later for apoptosis induction using a combination of propidium iodine (PI) and Annexin V double staining and flow cytometry. Double-negative cells represent the viable population (indicated by the percentages). (C) Reduced content of p53 favors the outgrowth of mature T cells derived from human CD34⁺ precursors. T cells were isolated from spleens and PB of mice reconstituted with transduced human CD34⁺ cells and were expanded in vitro, as described in “Materials and methods.” The percentage of GFP⁺ cells was established by flow cytometry at 2 different time points after stimulation.