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Help with Producing lentivirus in 293FT cells - Using Invitrogen's BLOCK-iT Lentiviral ol II miR RNAi Expression S (Apr/26/2007 )

During production of lentivirus in the 293FT cells, we observed high GFP in > 90% of cells, as well as "ballooning" morphology of the cells. This is right before harvesting at about 72hours.
(Protocol by Invitrogen followed strictly)

After harvesting the lentivirus, we would do a titration in the P19 cell line (also tried in 3T3 cell line) but none of the dilutions (1x, 100x to 10^6 x) were successful, we did not see any GFP at all.

Any ideas on why this might be so?

What time post transfection would you typically harvest the lentivirus?

Thanks! smile.gif


72 hours maybe a little long.

Usually, we collect the supernatant at 24 and 48 hours, and then test each fraction. Since the viruses are not stable at 37, they may be destructed at 72 hours.