The lentiviral vector is a pseudotyped virus, and its virulence gene is replaced by a foreign target gene, which only has the ability to infect once and cannot replicate to produce a new virus. Lentiviral packaging (the target gene expression vector, envelope plasmid, and packaging plasmid are transfected into 293T cells, and the virus is packaged in the cell, and then the virus supernatant is collected, and the required lentivirus is obtained after concentration, purification, and titer detection, which can be used to infect target cells.)
1. Lentivirus packaging steps.
6.Virus concentration purification: Viral particle-containing medium is harvested and subjected to 0The 22 m filter membrane was filtered and collected in a sterile ultracentrifuge tube, the supernatant was removed by ultracentrifugation, the precipitate was resuspended and the suspension was collected, filtered and sterilized again, and aliquoted in a sterile virus tube according to the amount of each use, and stored at -80
7.Titer: 293T cells with good growth status were diluted to 1 105 cell ml after digestion and counting, and added to a 96-well plate % CO2 incubator overnight. Make a 10-fold serial dilution of the virus for 6 consecutive dilutions and incubate overnight. The next day, aspirate the virus-laden culture and add 100 l of complete culture per well. Fluorescence is observed, cell counts are performed on wells with a fluorescence ratio of 10-30%, and the titer is calculated.
The calculation formula is as follows:
Titer (tu ml) = number of cells Percentage of fluorescence 103 volume of viral progeny (L).
2. Precautions.
1.Packaging cells.
As a commonly used packaging host, the health status of 293T cells is a key factor affecting viral yield. The passage number of 293T cells used for lentiviral packaging should not be too high, and 293T cells within 30 passages should be used.
2.Transfection efficiency.
Use optimized transfection systems and specialized transfection reagents to improve transfection efficiency.
3.Insert gene fragment size.
Lentiviral vectors have limited ability to accommodate the length of exogenous genes. Exceeding 3 kb can result in a significant reduction in packaging efficiencyPlasmids that are too large can also lead to reduced transfection efficiency, which in turn can affect the titer.
4.The timing of the virus.
The 24th and 48th h after transfection is the peak of lentivirus production, and the first batch of lentivirus can be harvested. At 48 h and 72 h, the titer decreased, and 1 2 lentiviruses could be harvested using a small volume of medium and combined with the first harvested lentivirus. If you are simply looking for high titers, you can harvest them once. In fact, the first harvest of lentivirus can meet the needs of multiple experiments.
5.Preservation of lentivirus.
1) Preservation conditions.
Store in -80 refrigerator;When using the virus, remove and place 4 cells after thawing for infection.
2) Avoid repeated freeze-thaw cycles to reduce viral titers.
Each freeze-thaw will reduce the viral titer by about 10% to 20%;In order to avoid repeated freeze-thaw cycles, it is recommended to aliquot according to the amount used each time.
3) When it is necessary to dilute the lentiviral titer, take out an appropriate amount of virus from the -80 freezer, put it in 4 to thaw, dilute it to a suitable titer with serum-free cell basal medium or 1 PBS (no Ca2+ Mg2+) for cell infection experiments, and store the diluted virus 4 as much as possible within three days, it is not recommended to carry out sub-packaging and cryopreservation.