TransIT-VirusGEN^® Transfection Reagent

Fig. 1 | 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), Competitor B4 (3:1), Competitor B5 (3:3:1) 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.

Fig. 2 | 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), Competitor B4 (2:1 (A) or 3:1 (B)), Competitor B5 (2:2:1 (A) or 3:3:1 (B)) 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.

Fig. 3 | em>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.

Fig. 4 | 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