Figure 1.

Testing of dual promoter lentiviral constructs for simultaneous expression of two genes. (A) Schematic of five lentiviral constructs used to infect 293 cells. The activity of three different promoters (EF1a, PGK, or CMV) is compared based on expression levels of gene position 1 (dsRed). Gene position 2 expression (green fluorescence protein [GFP]) is driven by the human ubiquitin C (UBC) promoter in each vector. (B) Flow cytometry analysis of 293 cells 3 days after infection with the lentiviruses shown in A. Multiplicity of infection (MOI) = 0.5 is employed to achieve an average single copy integration of each vector in approximately 1/3 of cells; at this MOI, approximately 2/3 of cells are predicted to remain untransduced (29). Only the combination of a cytomegalovirus (CMV) promoter in position 1 followed by a UBC promoter in position 2 effectively expresses both dsRed and GFP reporter genes in transduced cells. Cells transduced with CMV-dsRed alone or CMV-Mock-UBC-GFP are used as single color controls. Results are representative of experiments repeated three times at varying MOI.