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Question about transduction of lentivirus vector into fibroblast ? - transduction (May/13/2011 )

Now i have construct a lentivirus EGFP-fused vector, the fluroscence of EGFP signal can be observed when the vector was co-infected with virus-packing helper plasmid including pCMV and pMDG.The protocol list as following: After the lentivirus and it helper plasmid were infected in 293T cells with Fugene HD transfection reagent, next day, i replenished the 293T culture medium with 1% BSA and 8ug/ml polybrene (final conc.) containing complete (10% FBS) DMEM medium and incubate for 2~3 days. Next, the culture supernatant was then harvested and centrifuged for transfection to fibroblasts. I also added polybrene (final conc. 8ug/ml) in viral supernatants too, but i CANNOT observe the fluorescent EGFP expression in fibroblasts. Why? Can anyone tell me where i do wrong in this experiment? Oh My god, i repeat more than 10 times but the results still the same?


What is your centrifugation speed and time? Can you observe a pellet?
How about your supernatant, can you infect fibroblasts with it alone?