piggyBac/AAV transduction and colony formation assays. (a) Schematic representation of the piggyBac transposon expressing mCherry and the puromycin resistance gene, puromycin N-acetyl-transferase (Puror), driven by an RSV promoter and delivered by AAV2/5. Hyperactive piggyBac transposase (iPB7) is delivered in trans by an Ad5 vector. ITR, inverted terminal repeat of AAV; TR, piggyBac terminal repeat. (b) HeLa cells were cotransduced with increasing vector multiplicity of infections as indicated. Cells were selected for 10–14 days with 0.5 µg/ml puromycin, stained with methylene blue, and the drug resistant colonies were counted.