Lentivirus Production

Summary

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Abstract

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

Protocol

Part 1: Transfection of HEK 293 Cells To Produce Lentiviruses

*24 hours before transfection, plate 2.5 X 10⁶ cells in a 10cm dish for a confluency of 50-70% the next day.

1. Start this protocol by preparing the DNA with which one will later transfect one's cells to make virus. First, dilute FuGENE 6 Transfection Reagent Roche, a lipid reagent that make cells take up DNA. In a 1.5 ml tube, pipette 30μl FuGENE 6 into 600ul serum-free medium (SFDMEM). Be careful to not let FuGENE touch the side of the tube as it is delivered into the medium. Let this FuGENE and SFDMEM mix and incubate at room temperature for 5 minutes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM HEK 293T cells Penicillin-streptomycin Glutamine Phosphate-buffered saline FBS	Invitrogen
FuGENE 6 transfection reagent pSico mCherry pMDL pRSV pVSVG	Roche Group
Bleach	Clorox
10-cm cell culture dishes 96 well cell culture dishes	Falcon BD	353003
Multichannel pipette	Rainin
Filtered pipette tips	Rainin
1.5ml Eppendorf tubes Syringe Syinge filter (0.45-μm)	Eppendorf
Ultracentrifuge tubes	Beckman Coulter Inc.
Tissue culture incubator at 5% CO2	Coulter
Beckman SW41T.I rotor
Beckman ultracentrifuge	Beckman Coulter Inc.
Fluorescent microscope	Coulter
FACS Array	Beckman Coulter Inc.