LentiGuide-puro GeCKO library using 3rd generation lentivirus system

I just wondering anyone try GeCKO lentivirus production using pMD2.G, pMDLg/PRRE and pRSV-Rev system? Apparently my institution does not allow 2nd generation lentivirus system, and therefore, I have to switch to 3rd generation lentivirus system.

I have tried to package the GeCKO using this system in 10cm dish with Lipofectamine 3000, and yield about 10^6 TU/ml lentiviruses. I am not sure this amount represent the library? or I should concentrate the lentivirus prior to screen?

Advice needed!


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Fuchsia Kelpieover 4 years ago

I haven't tried packaging the GeCKO library with the 3rd generation lentivirus system before, but to answer your other questions:

Typically I make 4 T225's worth of lentivirus for a genome-scale screen. Whether you concentrate the lentivirus or not depends on how easy your cells are to transduce. Some cell types that are difficult to transduce will require lentivirus concentration prior to transduction.

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