Transduction of 293T and MLP29 cells by HIV-based lentiviral (LT) and MLV-based retroviral (RT) promoter traps. (A) A schematic of the vector trap integrated within the first intron of a cellular gene. The expression cassette is placed in reverse transcriptional orientation with respect to the vector framework. Ψ indicates viral encapsidation signal, including the 5′ portion of gag gene (GA); RRE, rev responsive element, cPPT, central polypurine tract (LT only); polyA, polyadenylation signal; SD and SA, splice donor and acceptor sites. (B, left) Southern blot analysis of 293T cells transduced with LT, RT, MLV-based retroviral vector (R), and HIV-based lentiviral vector (L). Matched vector amounts were used that yielded an average of 1 integrated vector copy per cell. A standard curve of plasmid DNA was used to calculate the vector copy-number. DNA was digested with AflII and the filter probed for puromycin^R-GFP sequences. (Right) FACS analysis (dot plot) of GFP expression in transduced cells. The percentage and mean fluorescence intensity (MFI) of GFP-positive cells, and the vector copy-number per cell (calculated by Southern blot), are indicated for each cell culture. (C) FACS analysis of GFP expression in MLP29 cells transduced with the indicated traps and left untreated (dot plots on the left; the MFI of GFP-positive cells is indicated) or treated with puromycin (dot plots on the right, and histograms; the MFI of the total population is indicated). UC indicates untransduced untreated cells. (D) FACS analysis (dot plot) of 293T cells transfected with the indicated amounts of linear plasmid forms of each vector trap. The results shown are representative of at least 3 experiments performed.