Lentiviral Particle Production and Titration

Lentiviral plasmids encoding shRNA were obtained from Sigma-Aldrich. Each plasmid was transformed into One Shot Stbl3 chemically competent cells (Invitrogen) and purified by ZymoPURE plasmid Maxiprep kit (Zymo research). Lentiviral pseudoparticles were obtained after plasmid transfection of 293FT cells using Lipofectamine 2000 (Invitrogen) or TurboFectin 8.0 Transfection Reagent (Origene). To prepare Vpx-VLPs, 293T cells were co-transfected by Lipofectamine 2000 or TurboFectin 8.0 Transfection Reagent with the 5 μg pMDL-X, 2.5 μg pcRSV-Rev, 3.5 μg X4-tropic HIV Env, and 1 μg pcVpx/myc, as described previously with some modifications^{37 (link),38 (link)}. The medium was replaced after 6–12 h with fresh media with 1X Viral boost (Alstem). The lentivirus or Vpx-VLPs containing media was harvested 72 h after transfection and concentrated 80 times using Lenti-X concentrator (Takara Clontech) or Lenti Concentrator (Origene). LV particles were then resuspended in RPMI 1640 media without serum and stored at −80°C before use. Virus titer was determined by using Jurkat T cells and Lenti-X GoStix Plus (Takara Clontech).