Figure 3

mRNA/PEI availability for the in vitro and in vivo translation and functionality of V5PB transposase. (A) In vitro translation of V5PB mRNA uncomplexed or complexed with PEI (N/P = 5). In vitro translation of mRNA alone (V5PB), in the presence of 150 mM NaCl (V5PB-NaCl) or complexed with PEI (V5PB-NaCl + PEI). Proteins V5PB Tp and Menin were separated by SDS-PAGE before chemiluminescent detection using an antiV5-Horseradish Peroxidase antibody. Mock is a negative control without mRNA. Experiments have been done in triplicate. (B) Time course of cellular production of V5PB transposase in HeLa cells (V5PB Tp). Transposase expression was assayed at the indicated time points, and monitored by Western-Blot experiments as described in (A). Mock: negative control (untransfected cells). Experiments have been done in triplicate. (C) pBluescript plasmid (pBSK ITR5′Néo^RITR3′) harboring the transposable element used in this study. This vector was composed of the expression cassette encoding the neomycin phosphotransferase protein (Neo^R) flanked by the inverted terminal repeats (ITR5′ and ITR3′), the recognition sites of the piggyBac transposase necessary for the transposition step. (D) Transposition assays in HeLa cells. Cells were transfected with the mRNA encoding the V5PB transposase and pBSK ITR5′Néo^RITR3′. Transposition events involve the integration of the Neo^R gene, and thus the emergence of a resistance phenotype to G418. Positive cells were selected under antibiotic pressure for 15 days, and colonies were then stained and counted. Mock is a negative control (without V5PB mRNA) corresponding to recombination events. The figures indicate the number of colonies counted (mean value ± standard deviation of three independent experiments made in triplicate).