Dear all,
I want to generate lentivirus, so I transfected plenti-C-GFP vector, PCMV-VSVG and PCMV-dR8.2 in 293T cells. I collected the medium 48h after transfection and tested the function of these lentivirus by infection in C2C12 myoblasts. However, there was no GFP signal, which means that the medium contained no lentivirus. Therefore, I wonder if anyone knows when should I collect the medium after transfection. What's more, does anyone know if 293T cells will die when you generating lentivirus? I know that 293A cells will die when I generate adenovirus. Thank you very much.
I routinely generate lenti-virus with 293T cells. They do not die after the virus production.
How can you be sure there's something wrong with viral production and not the infection itself? Meaning there are viruses but they do not infect the cells, for some reason. You can eassily check this but infection new 293T cells and then check for GFP.
I collect my medium 48 hrs post transfection with very high yield. It could be something wrong with your vector, DNA quality, GFP vector/packaging plasmids ratio, etc. How much ug of each plasmid do you transfect? Do you change to fresh medium 24 hrs post-transfection?
What transfection reagent are you using? We use tradional clacium chlorid or Calfectin. Either way, the total amount of DNA is 5-7.5 ug.
Also, are you sure the plasmids are good? Have they been tested or verified after purification?
Check if your 293T cells are GFP positive. That's an indicative they are producing your lentivirus.
I change medium 6h after transfection and I collect the supernatant 48h after transfection. I also use antibiotic free medium, it always worked better for me.
In that case, either your packaging plasmids not good or there's something wrong with the cells you're trying to infect.
Do you use polybren when infecting with virus?
Were your packaging plasmids evet validated?
Dear Gang, I agree with the previous comments. Some cells are hard to infect. To make sure you are actually producing virus, infect other cells that are easy to infect; for example HeLa cells or 293 cells. Then check for positive GFP cells after 48 hours. I assume you are filtering or spinning down the debris out (debris can reduce titer) and using polybrene. Spin infections (even with adherent cells) can help the infection efficiency. Good luck.
Dear Gang - what about concentrating your viral supernatant before infection. If your title is very low this will help you figure out whether it's a transfection or a packaging problem. I do agree that, at least for me, viral infection using Polybrene worked out better than any other infection agent.
Best of luck
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