Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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TransIT®-Lenti Transfection Reagent

Ideal for Recombinant Lentivirus Production

Mirus Bio Virus Production
TransIT<sup>®</sup>-Lenti

TransIT®-Lenti Transfection Reagent

Lentivirus Production

Overview of Recombinant Lentivirus Particles and Infection of Target Cell.
(1) Producer cells (e.g., 293T) are transfected with 3-4 plasmids encoding the gene of interest, vesicular stomatitis G protein (VSV-G), and essential virus proteins (e.g., gag, pol, and rev). (2) Virus is assembled and released into the supernatant through budding with the producer cell plasma membrane resulting in an envelope decorated with VSV-G. The medium containing virus is filtered through a 0.45 µm filter to remove any cells. (3) Target cells are frequently transduced with recombinant lentivirus particles in the presence of a polycation to increase aggregation and uptake. The virus enters the cell and the capsid is uncoated revealing the RNA genome and viral enzymes. The viral RNA is reverse transcribed into DNA which is then integrated into the host genome. (4) Transcription and translation result in the production of the protein encoded by the gene of interest.

Transit Lenti Transfection Reagent Competitor Benchmarking

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™ (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.

Transit Lenti Transfection Efficiency

High Efficiency Transfection with TransIT®-Lenti Transfection Reagent.
Adherent 293T/17 cells were transfected in a 6-well plate format using the MISSION™ vectors (pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix) 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.

Transit Lenti Scalability of Virus Production

Lentivirus Production is Scalable.
Adherent 293T/17 cells were transfected in a 12-well, 6-well, or 100 mm plate format using the MISSION™ vectors (pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix 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.

High Transduction Efficiency Lentivirus using Transit Lenti

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 the MISSION™ vectors (pLKO.1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix powered by MISSION™). 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 35 mm 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.

Features

  • High Performance – Provides up to eight-fold higher functional titers
  • Simple Protocol – No media change required, single harvest
  • Animal-Origin Free – High performance reliability

Description

  • TransIT®-Lenti Transfection Reagent is designed to enhance delivery of packaging and transfer vectors to adherent HEK293T cell types and increase recombinant lentivirus production.
  • The packaging plasmids include the essential virus components such as structural (gag), enzyme (pol), packaging (rev), and envelope (VSV-G) components of the lentivirus genome. The transfer plasmid encodes the gene of interest flanked by long terminal repeat (LTR) sequences, as well as other essential components such as RNA packaging signal (Psi) and rev response element (RRE).

Overview of Recombinant Lentivirus Production

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
MIR6603 TransIT-Lenti Transfection Reagent 0.3 ml 125,00 PDF
MIR6604 TransIT-Lenti Transfection Reagent 0.75 ml 313,00 PDF
MIR6600 TransIT-Lenti Transfection Reagent 1.5 ml 495,00 PDF
MIR6605 TransIT-Lenti Transfection Reagent 5 x 1.5 ml 2152,00 PDF
MIR6606 TransIT-Lenti Transfection Reagent 10 x 1.5 ml 3958,00 PDF
MIR6620 TransduceIT™ Transduction Reagent 1 ml 26,00 PDF
MIR6610 TransIT-Lenti Transfection Reagent 0.1 ml sample 0,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in selected countries.