Lentiviral Particle Generation and Transduction

To generate lentiviral particles, 8 × 10⁵ 293 T cells in a 6-cm dish were cotransfected with 2 μg of HIV-1 packaging plasmid pCMVΔ8.2 R (Addgene number 12263); 0.5 μg of the pCMV-VSV-G plasmid (Addgene number 8454) expressing protein G from vesicular stomatitis virus (VSV-G); and 3 μg of one of the pUCHR transfer vector constructed as described in the previous paragraph. Transfection was performed for 6 h using Lipofectamine 2000 reagent (Thermo Scientific, USA) according to the manufacturer’s instructions, and then the culture medium was replaced. Supernatants containing PVs were harvested and cleared through 0.45-μm-pore-size filters at 54 h posttransfection. The 293T/CD4 or Raji/CD4 cells that we described previously (67 (link)) were first infected with PVs to express CCR5. After immunostaining with respective MAb and positive sorting, cells were transduced to express one of the gp41 peptides. Peptide-expressing cells were then sorted based on mClover fluorescence. Transductions were set up using different doses of PVs. Two to three days postinfection the levels of transduction were quantified by flow cytometry, and samples with less than 30% positive events (low MOI) were selected for sorting.