I am trying to do some lentiviral infections of melanoma cells, the 293 FT cells always express the protein expression pretty well, but not the target cells. I used the same backbone plasmid having other gene as a positive control and get pretty good expression. My plasmid doesn't have GFP, so I check always by western blot only. With my positive control showing above 80% green cells, I know my packaging plasmids are working good (using second generation packaging, plasmid is FG12 backbone). My protein of interest is a Protease, one of my hypothesis is might be protein is itself killing the cells, where LV is infecting, is this a possibility? Although 293 ft are surviving and showing good expression. Is there any way to check, if the protein is killing the cells? One
Hi Ethika,
Did you count the target cells after transduction with positive control and protease? If the target cells were transduced and killed by the enforced expression of protease, you would observe significant cell death or reduced growth rate compared to the control.
Protesase expression may be very tricky due to its possible effects on cell metabolism. First thing you need to check is if your cell is actually dying. Count your cell before and after transduction. If the overexpression of the protease is killing your cell, you could try a different plasmid with a weaker promoter, you would still manage to insert your protease into your desired cell line but not as much copies, circumventing the lethality phenotype. Another thing you need to check is the antibiotics in your culture medium. Are you using antibiotics to select your transduced cells?
I usually do multiple infections, i.e. 3 times for 6 hrs each with Polybrene. I plate the cells in equal number and start my infections at~ 30-40% confluency. When I let the cells grow for 3 days of infection + 2 more days to check the expression, The cell numbers wouldn't stay similar anyway. And also in the infection efficiency is too low, I might not notice any significant change in cell number. Just by eyes, I really don't feel significant dead cells, in the infected ones. I am not able to use antibiotic selection, as the cells I am using are pretty resistant for the antibiotic (Zeocin), I have in construct.
Thanks for all the detailed information. The positive control, I am using is the same backbone+GFP, but not my target protein, my target plasmid doesn't have GFP. The positive control, was just used to check all the helper plasmids and the procedure is fine, it has nothing to do with target protein. 80% efficiency, I got in producer cells and above 60% in target cells with positive control. I get good expression (WB) always in the producer cells, but not in the target cells. The backbone of plasmid is FG12, and as I get good expression in target cells, it seems like gene is present there. The last two possibilities suggested for LV Production or maturation can be true, but the question is how to check these possibilities, any suggestions???? Regarding the confluency of target cells, I start with, it works with my positive control so I think, it should work with target protein too, the only reason, I start with low confluency is, If I start with high number, the plate will get too confluent within the multiple (3) infections. I don't like to split the cells during the infections. But sure, I can try using high confluency, may be that can help. Still, any suggestions for checking the production or maturation of LV?
Hi, Ethika.Did you solve the problem? Recently I am suffering from the same problem. I am eager to know how you handle it?
Have you ever tried to use 293 T cell as the target cell and infect them with the very virus you made?
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