Functional therapeutic gene expression in human T cells by SB-mediated transposition. (A) NGFR expression in the SB-nucleofected T cells (solid line). A murine IgG1 isotype mAb was used (broken line). (B) Cell killing by 5-FC. Human T cells nucleofected with SB transposon DNA (2 × 105 cells/well) or CEM cells (104 cells/well) transduced with retrovirus expressing NGCD were exposed to different concentrations of 5-fluorocytosine (5-FC, Sigma) in 96-well flat-bottom plates for 5 days. A colorimetric assay kit (CellTiter 96, Promega) based on the conversion of a tetrazolium dye to formazan was used to quantitate the number of viable cells on day 5. Absorbance was measured at 565 nm using a microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT). CEM cells alone or transduced with retrovirus expressing NGCD were used as negative and positive controls (data not shown). The data represent mean and standard deviation of 10 replicates. This experiment was repeated twice with similar results. Similar results also were obtained with the plasmid pT2/NGCD (data not shown).
