TransIT-VirusGEN® Transfection Reagent

TransIT-VirusGEN® Outperforms Competitor Reagents in Suspension & Adherent AAV Cell Cultures. Suspension FreeStyle™ 293-F cells grown in FreeStyle™ F17 Medium (A) or adherent 293T/17 cells (B) were transfected with pAAV-hrGFP, pAAV-RC, and pAAV-Helper (1:1:1 ratio, 1.5 µg/ml, Agilent Technologies) with the following reagents: TransIT-VirusGEN® (2:1, vol:wt), Lipofectamine® 2000 (3:1, Thermo Fisher), Lipofectamine® 3000 (3:3:1, Thermo Fisher) or 25 kDa PEI (4:1, Polysciences). Harvested virus was used to transduce HT1080 cells and GFP expression was measured 48 hours post-transduction using guava easyCyte™ 5HT Flow Cytometer. Functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the standard deviation of triplicate wells.

TransIT-VirusGEN® Outperforms Competitor Reagents in Suspension and Adherent Lentivirus Cell Cultures. Suspension FreeStyle™ 293-F cells grown in FreeStyle™ F17 Medium (A) or adherent 293T/17 cells (B) were transfected with pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix powered by MISSION® (1:1 ratio, 1 µg/ml)  with the following reagents: TransIT-VirusGEN® (3:1, vol:wt), Lipofectamine® 2000 (2:1 (A) or 3:1 (B), Thermo Fisher), Lipofectamine® 3000 (2:2:1 (A) or 3:3:1 (B), Thermo Fisher) or 25 kDa PEI (4:1, Polysciences). 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. Functional titers were measured from virus dilutions with less than 20% GFP positive cells. The error bars represent the standard deviation of triplicate wells.

TransIT-VirusGEN® Enables Broad Scalability (AAV). Suspension FreeStyle™ 293-F cells grown in FreeStyle™ F17 Medium (A) or adherent 293T/17 cells (B) were transfected with pAAV-hrGFP, pAAV-RC, and pAAV-Helper (1:1:1 ratio, 1.5 total µg/ml, Agilent Technologies) using the TransIT-VirusGEN® Transfection Reagent (2:1 reagent-to-DNA ratio (vol:wt), Mirus) at the indicated volumes perculture vessel. Harvested virus was used to transduce HT1080 cells and GFP expression was measured 48 hours post-transduction by flow cytometry. Functional titers were measured from virus dilutions with less than 20% GFP positive cells.  Error bars represent the standard deviation of triplicate wells, flasks, or dishes.

Higher Transfection Efficiency with TransIT-VirusGEN® Over PEI for Lentivirus Generation. HEK293T/17 cells were transfected with lentiviral packaging plasmids and either (a) control RFP transfer plasmid or (b) targeting SPdCas9-RFP transfer plasmids using either TransIT-VirusGEN® Transfection Reagent (3:1 reagent to DNA ratio; vol:wt) or 25 kDa linear PEI (3:1 reagent to DNA ratio; wt:wt). Brightfield and RFP Images were taken 17-22 hours post-transfection.

Data courtesy of Zuzana Drobna, PhD (Dr. Keung's Lab), Department of Chemical and Biomolecular Engineering,  North Carolina State University