Producing Lentivirus in 293T Cells with Polyjet

Abstract

Lentiviruses were produced by reverse transfection of suspended 293T cells using 20 ug lentiviral vector, 10 ug pVSV-G, 20 ug pCMV-dR8.91, and 100 μl Polyjet (SignaGen) or Lipofectamine 2000 (Life Technologies) in 15 cm dishes. The 3 ml plasmids–Polyjet complex and 1.5 ml 293T cell suspension were mixed in 50 ml centrifuge tubes and shaken for half hour before transferred to dishes. Virus harvested 48 and 72 h after transfection was combined, concentrated 50-100-fold by centrifugation at 26,000 rpm for 90 minutes, and suspended in culture medium. 500-2,000 μl of concentrated virus was used to infect 1×10(6) cells in 500 μl culture medium in the presence of 4 μg/ml polybrene (Sigma-Aldrich) followed by gentle pipetting 25 times and shaking for 10 minutes in the hood.