VirusGEN^® GMP LV Transfection Kit

TransIT-VirusGEN® Reagent and the VirusGEN® LV Transfection Kit Outperform PEI-based Reagents with Multiple Lentivirus Packaging Systems. Lentivirus production using TransIT-VirusGEN® Transfection Reagent +/- the VirusGEN® LV Enhancer was compared to Competitor A3 (2:1 reagent-to-DNA ratio) and Competitor C1 (4:1 reagent-to-DNA ratio) using their recommended protocol. Lentivirus was produced by transfecting Expi293™ cells grown in Expi293™ Expression Media (Thermo Fisher Scientific) at 4 x 10⁶ cells/ml with two different LV vector mixtures (1 μg/ml, 2μg/ml) including: (A) 3rd generation vectors pALD-LentiEGFP-A transfer vector and pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A packaging vectors (3:0.5:0.5:2 DNA ratio, Aldevron) or (B) 3rd generation vectors pLKO.1-puro-CMV-TurboGFP™ transfer vector (MilliporeSigma) and ViraSafe™ Pantropic Packaging mix (pRSV-Rev, pCMV-VSV-G, pCgpV, CellBio Labs) at a 3:0.5:0.5:2 DNA ratio. The VirusGEN® LV Enhancer was added to the TransIT-VirusGEN® condition at 18 hours post-transfection. Virus containing supernatant was used to transduce 293T/17 cells and GFP expression was measured at 72 hours post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. Lentivirus functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the range of duplicate wells.

TransIT-VirusGEN® Reagent and VirusGEN® LV Transfection Kit are Compatible with Multiple Cell Lines and Media Formulations for LV Production in Suspension 293 Cells. Viral Production cells (293-VP) grown in LV-MAX™ Production Medium (ThermoFisher), Expi293™ cells grown in Expi293™ Expression Medium (ThermoFisher), or FreeStyle™ 293-F cells grown in FreeStyle™ F17 (ThermoFisher) were seeded at 4 x 10^6 cells/ml prior to transfection (2 ml/non-treated 6-well plate). Lentivirus was produced using the TransIT-VirusGEN® Transfection Reagent +/- VirusGEN® LV Enhancer (Mirus Bio, 3:1 reagent-to-DNA ratio (wt:vol)) according to the recommended protocol. Lentivirus was produced by transfecting 3rd generation vectors pALD-LentiEGFP-A transfer vector and pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A packaging vectors at a 3:0.5:0.5:2 DNA ratio (Aldevron, 25 μg/flask (1 μg/ml final concentration). The VirusGEN® LV Enhancer was added 18 hours post-transfection. Functional titers from virus containing supernatant was measured following transduction 293T/17 cells and GFP expression was measured at 72 hours post post-transduction using Guava® easyCyte™ 5HT Flow Cytometer. Lentivirus functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the standard deviation of triplicate wells.