Best ratio of 5 Plasmids for Lentivirus production - (Sep/05/2012 )

Dear All,

I am planning to produce lentivirus. I have cloned my gene on 14Kb lentiviral plasmid. Now I need to package with 4 other plasmids (VSVG, TAT, REV, GAG).

What is the best ratio for these plasmids to transfect into 293Ts? I read somewhere that I need to use 20:1:1:1:2 ratio (Plasmid with the gene: tat: rev:gag:vsvg) Has anybody use this ration before?

Also, I am going to use PEI to transfect 293Ts. Does anybody have any experience using PEI for lentiviral production? I usually use the ratio 1:3 (ug DNA: ul PEI). Will it work for lentiviruses?

Thanks all

We used to make lentivirus with 293 cells in 15 cm plates. The mix was 20 micro gram plasmid+3 micro gram VsVg+ 3 micro gram rev+ 4 micro gram gag pol+ 5 micro gram pAdvantage with 70 micro liter of Fugene HD (transfection reagent) and we used to let it stand for 30 minutes before pipetting onto the cells. We had tried several transfection reagents and Fugene seemed to give us the best transfection. The media was changed the next day and the sups were collected at 48 and 72 hours and spun down to get the viral particles.

Zodiac,
thanks for your reply. I used TransIt (1:3 ratio 3 being TransIt). It seems to give good transfection efficiency (~70% of cells expressing GFP) I used 3:1:1:1:1 (3 being backbone) for a 10 cm dish. I also filter it using 0.45 um filter. Is it possible that the virus get stuck in the filter?

We used the same filter. I don't think there is any chance that the virus would get stuck in the filter.

Though you might have a good transfection, the problem might be with the production and release of viral particles. You might want to try using new batches of the viral components. I remember one time, our production was getting really low and we had to try all different combinations and finally it worked once we got a new vial of one of the components (don't remember which) from another lab.

OK, here I am again with my lenti problems
I have the virus working. Well, with TransIt and the ratio of 5:1:1:1:1. I usually get 60% green cells one day after transfection. When I use 1ml of the sup to infect 293T cells in a 12 well dish, I usually get 10-20 green cells. I guess the titre is too low?!
Now it gets even worse! When I ultracentrifuge the sup (~30 ml after three collections) and use the concentrated virus to infect, I donot get anything green after 24h??!!!
This is very strange for me, I have tried two different speeds for ultracentrifugation. Nothing! I tried not to filter th esup prior to ultracentrifugation. Nothing!
This is giving me bad headache these days. The only thing I can think of, is because GFP is under UBC promoter, it is getting expressed so weak that I cant see under normal conditions. Or as zodiac said, something is wrong with my helper plasmids...
Any ideas? Please help me.