about the lentivector production - (Jan/16/2007 )

Dear all,
I am preparing the lentivector using PWPT-GFP or PWPT-luc + PCMVDR+envelope encoding plasmid(pcDNA3.1-Env).I used the PWPT-GFP or PWPT-luc + PCMVDR+ pcDNA3.1 as negative control. However, my first trial gave me very confusing results. Because GFP expression was also detected in the negative control at Day 5 of transduction, and the expression level was comparable to the positive control. When I checked the luciferase activity, the negative cells gave about half of the RLU compared with positive cells.
What I have to mention is that I didnot filter the supernatant. Instead, I did the centrifugation and used 250 microlliter supernatant as virus input(no concentration, no dilution). Did this step make difference? Besides, is there any posibility that the lipofectamine-DNA complex still remained in the sup and resulted in the positive signals in the target cells?If so, is the concentration step a must?

Looking forward to any suggestions.Thanks a lot.

Try filtering the supernatant in addition to centrifuging so that cells from transfection are not carried over during infection. But inspite of this u still find GFP positive cells in ur negative control, i would suggest to verify the plasmid by running them on gel or by appropriate restriction digest.

Dear scolix, I did the experiment again as you suggested. The supernatant was filtered through 0.45 um filter. In addition, I made 10 fold dilution to minimize the carry-over. After overnight incubation with target cells, the supernant was replaced with fresh medium. However, results did not become better. Negative control still gave positive signals (GFP expression) even after 100,000 dilution. I have checked the plasmids by enzyme digest and they seemed to be fine. And the expression of envelope protein was confirmed by IFA in plasmid encoding env gene, while not in empty vector. It really drove me crazy. Have you ever come cross such kind of problems? Look forward to your ideas, thanks a lot.

Try to omit the PCMVDR from the transfections for the negative controls.

May b this could solve the problem.

The thing is I dont do these negative controls. As only when I get good virus productin do I get any florescence in my cells upon infection.

Good luck!!!