T-cell development and transgene expression in SB100X-transfected CD34⁺ HPCs. (A) The percentage of DsRed⁺ cells after transfection of CD34⁺ HPCs with SB transposon alone or both transposon and SB100X transposase. Nucleofected CD34⁺ cells were cocultured onto OP9-DL1 stromal cells + Flt-3L and IL-7 and analyzed for DsRed expression by flow cytometric analysis on days 1, 7, 14, and 21. (B) T-cell development and phenotyping. Transfected CD34⁺ HPCs were cocultured with OP9-DL1 or OP9-GFP stromal cells for 28 days and assayed by flow cytometry for the expression of CD4, CD8, CD1a, and CD7. In CD34⁺ SB transposon + SB100X transposase-transfected HSCs, the DsRed⁺ cells were gated and analyzed for the expression of CD4/CD8 and CD1a/CD7.