Figure 1

Construction of the Rev-dependent lentiviral vectors. (A) Schematic representation of the Rev-dependent lentiviral constructs. Shown are pNL-GFP-RRE-SA and its derivatives. The HIV-1 5' LTR, packaging signal (ψ), splice donors (D1, D4) and acceptors (A5, A7), IRES, RRE, and 3' LTR are indicated. The Rev-dependent constructs would transcribe both spliced and unspliced transcripts as HIV-1 does. Only the unspliced or partially spliced transcripts that contain reporters or toxins are Rev-dependent for expression. α-HL is the α-hemolysin of Staphylococcus aureus. (B) Schematic representation of the HIV-1 helper construct, pCMVΔR8.2, in which both the viral package signal (Δψ)and the envelope gene (Env) were deleted. (C) Simultaneous expression of two genes via IRES in the Rev-dependent construct. Both the E. coli lacZ and the GFP genes were cloned into the vector, pNL-lacZ-GFP-RRE-SA, which was cotransfected with pCMVΔ8.2 into HEK293T cells. For comparison, pNL-GFP-RRE-SA was similarly cotransfected. Shown is GFP expression measured by flow cytometry at 48 hours post cotransfection with either pNL-GFP-RRE-SA (middle panel, GFP) or pNL-lacZ-GFP-RRE-SA (right panel, LacZ-GFP). (D) Cells from pNL-LacZ-GFP-RRE-SA cotransfection were also stained for LacZ and visualized under the microscope for LacZ and GFP. One hundred cells were counted, and among them, 20 expressed both LacZ and GFP; 4 expressed only LacZ; and 76 expressed neither LacZ nor GFP.