I am trying to establish a lentivirus system in a new lab. Purchased pMuLE lenti destination vectors, psPAX2, and pMDG.2 from addgene and 293T cells from Dharmacon. First batch of virus tittered by selection/cell survival very high but did not check for expression. Moved forward to make constitutive cell lines and got neomycin resistance but no expression of my GOI. Made a control GFP virus (sorry, didn't look at producing cells) that does not give me any GFP positive 293T cells after transducing. Transfection of the plasmid, however, does! The transfection for virus is done with a lipid-based reagent and a 4:3:1 ratio, target:package:envelope. 72hours after the transfection, cells are multinucleated and look fantastic for viral production. I centrifuge, filter (0.45um), and aliquot for freezing. I add polybrene after thaw for transduction and well as pretreat target cells for one hour prior to viral infection.
Can anyone help with why I'm getting selection resistance but not expression???
It is a bit strange that the cells are not at all positive cell after transduction specially if the same plasmide get positive cells after transfection. We had something similar in the past. I strongly recomment the type/generation of your lentivirus production. You can not mix second with third generation production method. the Tat protein for encapsulation is not produced similarly between the generation. Hope it helps
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