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Lentivirus vector – Production/Packaging Protocol, Guidelines And References
Lentivirus Production Protocol--Genemedi
1. Lentivirus plasmid construction
The gene of interest is cloned into one of the LTR/MCS-containing lentivirus vectors to generate pLV-GOI. The purity and RNA contaminants of viral plasmid should be taken into consideration.
2. Lentivirus packaging
The recombinant lentivirus viral plasmid pLV-GOI is co-transfected into the 293T with envelope expressing plasmid pMD2G and packaging plasmid pSPAX2, which together supply all the trans-acting factors required for lentivirus replication and packaging in the 293T cells.
3. Harvesting virus particles
Collect the supernatant containing lentivirus particles 48 hours and 72 hours post transfection, respectively. Replace fresh the culture medium after the collection of supernatant at 48h.
4. Virus purification
After collecting virus twice, discard the transfected 293T cells and filter the collected supernatant with 0.45μM filter membrane to an ultracentrifuge tube. Centrifuge at 72000g for 2 hours at 4℃. Then discard the supernatants and resuspend the lentivirus deposition with 500μl fresh medium and keep at -80℃ or in liquid nitrogen for long time storage.
5. Lentivirus titer detection
Determine lentivirus titer with fluorescent microscopy. Seed 293T 1×104 cells/well in a 96-well plate one day in advance and perform gradient dilution of the lentiviral particles to 1:10, 1:100, 1:1000, 1:10^4, 1:10^5, 1:10^6 in 100ul final volume in culture medium. Total 100ul viral particle mixture should be added to each well with at least 3 replicates per virus. Two days post infection, count the fluorescent positive cells using fluorescent microscopy and select the dilution factor with a proper fluorescent positive proportion (10%-30% positive cells/well). Count the triplicates and average the number of positive cells. Estimate the lentivirus titer using the following formulation: Viral titer (TU/ml) = number of fluorescent positive cells × 10 × dilution
6. Quality control of lentivirus
After lentivirus titer detection, the infection activity needs to be evaluated before animal experiment by infecting cells such as 293T, CHO to test the gene expression.
NIH Guidelines for Adeno-Associated (AAV) Lentivirus and Adenovirus