Hello all,
During the last few months I tried to make a lenti virus with my target gene. (I used this protocol by Addgene : https://www.addgene.org/protocols/lentivirus-production/). For the production of the virus I used psPAX2 and pMD2.G plasmids and PEI. In the beginning I purified the DNA plasmids by mini-preps (Although it was an exhausting process) and I got really nice results (~90% of infection). Later I began to use maxi-preps to upscale my work and for some reason, which I don’t know, I got a very low titer (Even though I see high transfection rate in the 293T packaging cells). What can I do to improve my virus production efficiency?
Hello Iris, you could use polyethelinimine-mediated transfection. There are quite a lot of factors that can affect viral production efficiency like vector, vector integrity, target gene, collection time, packaging cells, shRNA-mediated cleavage, transfection etc. Please follow the links attached.Article An improved method for increasing the efficiency of gene tra...
Article Optimization of lentiviral vector production using polyethyl...
Sometimes blind passage of the virus in the host cells improves the infectivity of the virus. Give a couple of passages and then use feeze thaw cells lysate for infection of host cells.
High transfection rate doesn't always identify good production (as your transgene plasmid can be efficiently transfected instead of the packaging plasmid). Any details on the viability of the cells during the harvest of the virus and the incubation time period of your transfection mix (DNA and PEI complex) before adding it to the cells.
As mentioned above, higher transfection doesn't guarantee high yield. I think that you have to start from DNA. Did you run the previous and current DNA on a gel to check whether the DNA are the same? If it is the same and If u still think the prob is with DNA try checking the virus production in small scale using both DNA batches. Meantime you can also check on the 293T condition, passage number now and then.
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