TransIT^®-Lenti Transfection Reagent

Fig. 1 | High Functional Titers with TransIT^®-Lenti Transfection Reagent. Adherent 293T/17 cells were transfected in a 6-well plate with pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix Powered by MISSION^® Genomics (1:1 ratio, 2 µg/well) with the following reagents: TransIT^®-Lenti (3:1, vol:wt), Lipofectamine^® 2000 (3:1), Lipofectamine^® 3000 (3:1:1), 25 kDa PEI (6:1), or CaPO₄ precipitation (4 µg pDNA/well). 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.

Fig. 2 | Comparison of CaPO₄, Lipofectamine^® 2000 or TransIT^®-Lenti Generated Lentivirus. HIV CMVeGFP Virus was produced in HEK 293FT cells using either CaPO₄, Lipofectamine^® 2000 or TransIT^®-Lenti Transfection Reagent per the manufacturer's protocol. Lentivirus was collected 48 hours post-transfection and concentrated by prolonged centrifugation at 9,000 x g. HT1080cells were infected with a 1:100 or 1:1000 dilution of each concentrated lentivirus. Images were captured 48 hours post-transduction. Data courtesy of Jeremy Coffin, University of Iowa Viral Vector Core

Fig. 3 | High Transduction Efficiency with Unconcentrated Lentivirus Using TransIT^®-Lenti. (A) Lentivirus was produced with the TransIT^®-Lenti Transfection Reagent (3:1, vol:wt) or Lipofectamine^® 2000 using pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix Powered by MISSION^® Genomics. The supernatant was harvested, filtered (0.45 µm), and frozen. Lentivirus transductions were performed 5 days post-plating with iCell^® Motor Neurons (Cellular Dynamics International). For both TransIT^®-Lenti and Lipofectamine^® 2000, one microliter of unconcentrated supernatant was added per well of a 96-well plate. GFP efficiency was measured 72 hours post-transduction using guava easyCyte™ 5HT Flow Cytometer. Error bars represent the SEM of duplicate wells. (B) iCell^® Motor Neurons were plated in ibidi 35mm dishes and transduced with lentivirus produced using the TransIT^®-Lenti Transfection Reagent and MISSION^® vectors. Images were captured at 72 hours post-transduction with a Zeiss Axiovert S100 inverted fluorescence microscope using a 63X objective under oil.

Fig. 4 | High Efficiency Transfection with TransIT^®-Lenti Transfection Reagent. Adherent 293T/17 cells were transfected in a 6-well plate format using the pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix Powered by MISSION^® Genomics using the TransIT^®-Lenti Transfection Reagent (3:1, vol:wt). GFP efficiency was measured at 48 hours post-transfection using guava easyCyte™ 5HT Flow Cytometer. Error bars represent five transfection complexes. Images were captured at 48 hours post-transfection (10X objective) using a Zeiss Axiovert S100 inverted fluorescence microscope. The observed cell rounding and cell-cell fusion is due to high expression of the vesicular stomatitis virus G protein (VSV-G) for pseudotyping the recombinant lentivirus.

Fig. 5 | Lentivirus Production is Scalable. Adherent 293T/17 cells were transfected in a 12-well, 6-well or 100 mm plate format using the pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix Powered by MISSION^® Genomics at a 1:1 ratio, and 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.