Packaging Steps and Experience Summary of Lentivirus

Abstract

Lentivirus is a one of retrovirus. It can introduce target gene into some difficult-transfected cells, such as primary cells, and integrate target genes randomly into the host genome, thereby greatly increasing the transfection efficiency, with stable expression in cell lines for several generations. It can also be used for screening stable cell strain. Because there is a random integration with uncertain factors, some companies provide targeted integration technology to integrate target gene into the genome of the specific position, so as to ensure the high expression and not to generate damage to cell produced by random integration.

Content

The processes that lentiviral vectors contain packaging, transfection and stable genetic information integration. Lentiviral packaging plasmids can provide all auxiliary proteins needed in transcription, packaging and recombination. In order to produce virus particle with high titer, it needs to use the expression vector and packaging plasmids to co-transfect cells, and do virus packaging in cells. The purified virus particles are secreted into the extracellular medium, and the supernatant can be directly used for host cell infection after centrifugation.

Lentivirus genome is a positive stranded RNA. When the genome goes into cells, it is transformed into DNA by reverse transcriptase owned by itself and forms pre-integration complex to enter the nucleus. DNA is integrated into the cell genome. The integrated DNA transfers mRNA, and returns to the cytoplasm, expressing target protein or producing RNAi interference.

The lentivirus packaging system consists of a packaging plasmid mixture (Mix) and a lentiviral vector plasmid.

The vector contains the basic components of HIV, such as 5 ‘LTR, 3’ LTR and other auxiliary components.

Packaging plasmid mixtures are not the same in different systems. The three-plasmid system in the laboratory is taken as an example. The plasmid mixtures contain pMDL, VSVG, pRSV-Rev, and the ratio in 5:3:2. Among them, pMDL contains the gag gene encoding the major structural protein of HIV virus and the pol gene encoding the virus specific enzyme; PRSV-Rev has a regulatory factor rev gene that regulates the expression of gag and pol genes; VSVG contains the VSVG gene that comes from the herpes simplex virus needed for viral packaging.

Following is the introduction of 293T cells in 6-well plate (35mm) for viral packaging. the other plate should be regulated corresponding to volume.

Preparation

Reagent chapter

Plasmid

293T cells with exponential growth

Virus packaging plasmid Mix:1 µg/µl (Mix=pMDL: VSV-G : REV=5:3:2): Packaging virus plasmid is different by different vector systems. This system can be used for packaging PBOBi, PLKO and Plv plasmid

Transfection reagents: turbofect or lipo2000

Cell sorting

Cells were collected by trypsin digestion, and cells were cultured on the 35mm culture dish with suitable complete medium (According to the experiment, the culture dish was selected, and the area occupied reached more than 80% of the total area of the culture dish). The cells were incubated in 37℃ box containing 5% CO2. When the cells were completely adherent, they could be transfected. 2 h before transfection, 1.5ml complete-medium was replaced with the old medium.

The mixing of core plasmid and packaging plasmid

Take a 1.5ml EP sterile tube, and add 400 µl serum-free DMEM, 1.5 µg core plasmid and 1.5 µg virus packaging plasmid, 6 µl turbofect (turbofect: plasmid = 2:1), mixing them and and keep them static for 15–20min.

Incubation

The mixture of this 400 μl was added into the cell culture medium of above monolayer, gently shaking the plate after mixing and keep it for incubation in 5% CO2 box.

Collecting virus supernatant

After 6–10 h later, suck away the medium and add the 2.5 ml 37℃ complete-medium. Cells continue being incubated. About 40 h later, lentivirus supernatant can be collected to infect or to be packed in －80℃ for use.(by 1500rpm centrifugation for five minutes, we can collect 2ml supernatant containing virus.)

Virus infections

1. The cells are spread in six-well-plate or twelve-well-plate. The best density is 30% 70%.

2. For six- well-plate (twelve-plate is half):

Infectmouse cells：800–1600µl virus + 1600–800 µl fresh medium =2.4 ml totalInfecthuman cells：200–800µl virus + 1200 µl-800 µl fresh medium =2 ml total

3. Add 2–5 g/ml polybrene.

4. After 12–24 h, change the fluid and do experiment.