TransIT® Lentivirus System

Up to 100-fold Increase in Functional Lentivirus Titers. Adherent 293T/17 cells were transfected in a 6-well plate with the pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix Powered by MISSION® Genomics or pLenti6.2-GW/EmGFP transfer vector and the ViraPower™ Lentiviral Packaging Mix (1:1 ration, 2 µg/well) with the following reagents: TransIT®-Lenti (3:1, vol:wt), Lipofectamine® 2000 (3:1), or Lipofectamine® 3000 (3:1:1). The supernatant was harvested, filtered (0.45 µm), and titered using 293T/17 cells. Lentivirus transductions were performed in the presence of 8 µg/ml TransduceIT™ and GFP expression was measured 72 hours post-transduction using guava easyCyte™ 5HT Flow Cytometer. Error bars represent triplicate transfection complexes titered individually. Functional titers were calculated using virus dilutions with less than 20% GFP positive cells.

Transfer Vector Selection Greatly Affects Functional Lentivirus Titers. Adherent 293T/17 cells were transfected in a 6-well plate with one of three transfer vectors: MISSION® pLKO.1-puro-CMV-TurboGFP™, ViraPower™ pLenti6.2-GW/EmGFP, or Precision Lenti-ORF™ RFP and the Lentivirus Packaging Mix powered by MISSION® Genomics) (1:1 ratio, 2 µg/well) using the TransIT®-Lenti Transfection Reagent (3:1, vol:wt). The supernatant was harvested, filtered (0.45 µm), and titered using 293T/17 cells. Lentivirus transductions were performed in the presence of 8 µg/ml TransduceIT™ and GFP expression was measured 72 hours post-transduction using guava easyCyte™ 5HT Flow Cytometer. Error bars represent triplicate transfection complexes titered individually. Functional titers were calculated using virus dilutions with less than 20% GFP positive cells.