Figure 1.

Construction of a lentivirus-based vector for delivering anti-human CCR5 siRNAs. (A) Schematic diagram of the siRNA-expressing lentiviral vector. The short hairpin form of siRNA is expressed under the control of a human U6-RNA Pol III promoter (Pol III). The vector also contains a human UbiC promoter driving the GFP marker gene for tracking transduced cells. 5′ LTR, HIV-1 5′LTR; Δ3′ LTR, HIV-1 self-inactivating 3′LTR; Flap, HIV-1 DNA flap element; WRE, woodchuck hepatitis B virus RNA regulatory element. (B) Selection of siRNA constructs that can effectively inhibit CCR5 expression in Magi-CCR5 cells. Magi-CCR5 cells were transduced with various lentiviral vectors. The cells were harvested 4 days after virus transduction and analyzed by FACS with anti-human CCR5 or isotype control antibody staining. The productively transduced (GFP+) and nontransduced (GFP−) cells were gated based on their GFP signals. The CCR5 staining is represented by the open curve and the isotype control by the shaded curve. Note that some lower GFP-expressing cells were included in the GFP− gate and, thus, there appeared to be a slight decrease of CCR5 staining in this population of the anti-CCR5 vector-transduced samples. CCR5-siRNA(186), the vector expressing a potent anti-CCR5 siRNA; CCR5-siRNA(809), the vector expressing a weaker anti-CCR5 siRNA; lacZ-siRNA, a nonspecific siRNA vector; FG12, empty vector; Mock, mock transduction control.