Fig. 7

IT is transpositionally active in mammalian cells. a IT and Sleeping Beauty (SB) transposon vectors for in vitro transposition activity assays. SB was used as a positive control. The three TIR vectors—pITo-Neo, pITc-Neo and pSB-Neo—have the same vector frame, and the TIR elements on both sides are the only differences. pITo-Neo is composed of two original TIR sequences of IT, pITc-Neo is composed of one consistent TIR and the other original TIR of IT, and pSB-Neo is composed of two SB TIRs. The two transposase vectors pCMV-itTPase and pCMV-SB100X also have the same frame. b–e HeLa cells were stably transfected with 1 μg of mixed plasmids (donor and helper plasmids at a 1:1 ratio. For selection, the transfected cells were reseeded onto 10-cm dishes (b and c, 1:100 plating; d and e, 1:10 plating). Selection was performed using 1000 mg/ml G418 for 14 days in DMEM. To determine the relative rates of transgenesis, foci of neomycin-resistant cell colonies that remained on each dish were counted after being fixed in 4% paraformaldehyde and stained with methylene blue. Bars represent the mean neomycin-resistant colonies ± standard deviations from three independent experiments