FIGURE 6.

PiggyBac can stably transfer large genes. IL-15-treated PBMCs (from 4 healthy donors) were co-transfected with 5.7 μg pCMV-PB and 5 μg pIR-eGFP (5.7 kb transposon in a 7.2 kb vector), plus 5.6 μg pUC19 (as filler DNA) or 10.6 μg pIR-mTor^HA-eGFP (13 kb transposon in a 15 kb vector) and stimulated with OKT3 (50 ng/mL) on feeder cells in the presence of IL-15 (10 ng/mL). The percentage of transfected (GFP+) cells (A) and total cell number (B) was determined at 1 and 8 days post-transfection by FACS analysis and counting by trypan blue exclusion, respectively. Transfected cells were enriched in each culture to >80% after sorting for GFP+ cells. Sorted GFP+ cells from the pIR-eGFP and pIR-mTor^HA-eGFP transfections expanded similarly and rapidly after stimulation with OKT3 on feeder cells and maintained GFP expression (shown in parentheses above each data point) (C). Mock transfected cells were subjected to the transfection procedure without DNA and were not sorted. Expansion data is representative of 3 healthy donors. D is a western blot showing the expression of mTor^HA in transduced T cells using an antibody to the HA tag.