low expression efficiency using 293T to produce retrovirus - does Na pyruvate matter for 293T growth ? (Aug/28/2006 )

Hello,

I've tried to co-trasfect 293T (10cm dish) with 3 plasmids, which containing gag/pol, vsvg and the retro-expression vector (with GFP). I used Lipofectamine 2000 as the trasfection reagent and either the propotion of the 3 plasmids or the DNA/liposome ratio has been optimised. GFP can be detected after 24h onwards using fluorescent microscope (GFP getting stronger by days), and the supernatant were collected at 48h onwards till cells died. However when I tried to titrate the virus titer on 3T3 at different time point, all gave me very low titer at around10^3-10^4/ml. I am not sure where I got wrong, doesn't the stonger GFP under microscope mean higher virus concentration ? Or it could be false positive, which means though GFP is shining, the virus could still be incompletly produced (due to the incomplete integration of the 3 plasmids ?) ? Can someone with experience provide me advices on how to improve it ?

Many thanks.

I collect virus first collection) 36 hrs after transfection and then a second collection 24 hrs from the first collection. Dont let the virus stay in the media for lond as they have a half life fo some 8-10 hrs and get degraded the longer they r in the media.

U must b getting good transfection as u c GFP shining. But this doesnt gurantee a good titre. U need to opitmise ur vector and u could centrifuge the supernatant u collect from the 10cm plate to concentrate it and increase ur titre. I am getting more than 1 x 10^8.

Also some people use cholesterol and sodium butyrate to increase virus production. And one could also use polybrene before infection ur cells, improves infection.

What ratios of the 3 plasmids r u using.

thanks Scolix, i did use polybrene to improve my infection to 3T3. I am not aware the time of virus staying in the medium could degrate its titer, however when trying to optimise the time point, I collect every 24h and replace with fresh medium. The ratio of the 3 plasmids used in 10cm dish is vsvg/gagpol/MIG-w : 15ug/10ug/10ug. some collegue told me 1:3:4 gave good retuls in 6 well plate, but I haven't tested yet.

I use a ratio of 1:1:1 , and I find it ok. But some people claim to vary ratios to get better titres. I havent got the time to try out these ratios.

If u can modify ur vector my adding cPPT sequence, this increases titres by many fold.

thanks Jusu, when you said store the virus properly, do you mean by growing virus in more plates/dishes and collect the virus supernatant at the right time point at once ? do you usually pool it all together ? how about concentrate it using PEG/NaCl ?

another concern just occurred to me is that does the culture condition of 293T affect ? we know 293T grows in DMEM/10%FCS, but does the existance of Na pyruvate matter ? just found that some DMEM powder contains NO Na pyruvate and some did. the one I used is with high glucose (45oomg/L) and L-glutamine, but without Na pyruvate.

i usually add Na-pyruvate and Non-essential amino acids to the media.

I concentrate by spinning the supernatant at 100,000x g.

store virus in -80C