Lentiviral Vector Production and Quantification

HIV-1 NL4-3, NLENY1, and YU-2 were packaged in 293T cells with native HIV-1 envelope or pseudotyped with VSV-G envelope. Viral packaging was done with 10–17 µg of each lentiviral construct and 3–4 µg of VSV-G plasmid DNA, using Lipofectamine plus or Lipofecamine-2000 (Invitrogen) as described earlier [7] (link),[18] (link),[59] (link). After 16 h of transfection, cells in 100-mm dishes were washed twice with fresh medium and incubated in RPMI 1640 containing 10% FBS. Culture supernatants were harvested 72–80 h after transfection, centrifuged at 1,200 rpm for 15 min, and filtered through a 0.20 µm filter. The filtrate, after the addition of MgCl2 (4 mM), was digested with 10–50 units of RNase-free DNase (Invitrogen) per 1 µg of plasmid DNA for 30 min at 37°C, then aliquoted and stored at −80°C. The viral stocks were quantified for p24 antigen by ELISA (ZeptoMetrix). A viral inoculum of 50–1,000 ng/mL was used to infect lymphocytes, HFA, and SVGA.