I need to package lentivirus to generate gene of interest stable brain tumor cell line so, I want to know what is the required concentration of packaging, envelop and transfer vectors to transfect the HEK-293FT cell line? and at which time of interval media need to be collected from transfected cells. I would highly appreciate if someone can share detailed protocol what they are using in their lab.
That depends on which Env you`re going to use to deliver your lentivector particles to target cells. If we are looking at VSV-G protein (most often), many protocols suggest the plasmid ratio of 3:2:1 for vector:packaging:VSVG expression plasmids. I`d suggest 15 mcg of vector, 10 mcg of helper, and 5 mcg of pCMV-VSVG (or similar) for a 60 mm dish, transfected by calcium phosphate-DNA precipitate. If you wish to use the HIV gp16o as Env, you will probably take similar or twice as much DNA of this plasmid, as compared to the vector DNA (as gp160 is poorly expressed on the cell membrane and poorly incorporated into the vector particles)..
You will collect about 70-80% of infection titer 24 h post-transfection, and the rest at 48 h p/t, the residual infectivity (72 h) is usually negligible.
Dear Sergey, Thanks for the articulate answer. I followed the same ratio what you have mentioned. Thanks for kind help.
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