FIG. 1.

Determination of the optimal ratio of HSV-HSB5 to HSVT-βgeo amplicon to achieve maximal SB-mediated transposition in vitro. (A) Schematic representation of the amplicons used in the in vivo testing of the HSV/SB amplicon vector platform. The HSVPrPUC amplicon contains a multiple cloning site, and serves as an empty vector control. The HSV-SB10 amplicon expresses the “wild-type” SB transposase, whereas the HSV-HSB5 amplicon expresses the “hyperactive” HSB5 transposase. HSVT-βgeo serves as a substrate vector for the transposase, and contains an IR/DR-flanked Rous sarcoma virus (RSV) promoter-driven β-galactosidase–neomycin phosphotransferase fusion reporter gene unit (β-geo; adapted from Bowers et al., 2006). (B) Assessment of the optimal transposase-to-transposon (HSV-HSB5:HSVT-βgeo) amplicon ratio required to achieve maximal transposition efficiency. HEK 293 cell monolayers were individually cotransduced with various ratios of HSV-HSB5 and HSVT-βgeo viral particles at an MOI of 0.5. HSVPrPUC was used as a “balance” virus to ensure that equal numbers of viral particles were transduced under each condition. Two days posttransduction, the cells were seeded onto 100-mm dishes at a 1:3 dilution and placed under G418 selection for 2 weeks. G418-resistant colonies were enumerated by X-Gal histochemistry. Error bars represent the standard error of the mean and statistical analysis was conducted by Student t test. Color images available online at www.liebertonline.com/hum.