Best ratio of 5 Plasmids for Lentivirus production
Posted 05 September 2012 - 03:17 PM
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?
Posted 18 October 2012 - 06:19 AM
Posted 18 October 2012 - 10:27 AM
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?
Posted 18 October 2012 - 11:31 AM
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.
Posted 16 February 2013 - 10:57 PM
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.