help with retrovirus production - have you ever produced retrovirus by yourself? (Mar/17/2006 )
Hi, everyone,
i transfected 293T cells with pVPACK-GP, pVPACK-AMPHO and pFB-NEO-LACZ using FUGENE6. but i didn't get any retrovirus---i didn't saw any blue cells by doing x-gal analysis after transduce a few human tumor cell lines with the supernatant of transfected 293T cells.
by the way, i have also tried the transfection protocol of MBS kit from stratagene in vain.
could anyone with the experience of producing retrovirus by himself help me out?
thanks in advance.
I've been producing HIV from 2 plasmids (or plasmid combined with PCR-product) by myself on 293-T cells, as well as producing hiv on 293-T starting from a plasmid with the entire viral genome in there.
All approaches work (and are done for different reasons), but I noticed that co-transfecting 2 plasmids (or plasmid and PCR product) leads to lower viral titers (which makes an awful lot of sense, as each cell that is producing virus has to have been transfected with the 2 parts instead of only 1). So I fear that maybe due to the fact that you are transfecting 3 plasmids, that this might result to even lower yields, maybe undetecable low? Is there any possible way of co-transfecting 3 plasmids with a reporter gene in there (for instance EGFP, DsRED2 and maybe LacZ, so you might see if you get triple transfected cells)?
Maybe it's also not a bad idea to make a cell line that is stably transfected with one of the three vectors, so you would afterwards only need to transfect 2.
i transfected 293T cells with pVPACK-GP, pVPACK-AMPHO and pFB-NEO-LACZ using FUGENE6. but i didn't get any retrovirus---i didn't saw any blue cells by doing x-gal analysis after transduce a few human tumor cell lines with the supernatant of transfected 293T cells.
by the way, i have also tried the transfection protocol of MBS kit from stratagene in vain.
could anyone with the experience of producing retrovirus by himself help me out?
thanks in advance.
well, i think that contransfecting is not an issue - it is "all or none". thats why people are cotransfecting GFP with their stuff to identify expressing cells.
i make viruses contransfecting 4 plasmids, and had no problems with that.
making a stable is not a bad idea - but it all depends. some viral proteins are toxic for the cells, and people prefer to contransfect insted of making stables.
for the titer - the more WT virus genes you have, the higher titer you'll get. im not familiar with the system you're using, but i think you should first make sure youre 293Ts are in good condition, and your transfection is efficient. plasmid ratio makes a difference too.
if all that works, there is another issue - some cell lines are extremely hard to transduce! maybe your titer is quite fine, but the cell line is a problem? maybe you can, as a control, infect some other cells, just to make sure that you have a virus in there? if yes, you can work on your infection procedure..