I usually use lentiviral vectors but there's some problems with toxicity/efficiency... Could you share your experience?
In the past, I have made viruses using the protocol by Tyler Jacks lab at MIT. They usually produce high titers and excellent gene overexpression in neurospheres. Do you think your gene of interest is causing toxicity to the cells?
Hi Jason, I'm not sure it's the gene because it was efficiently transducted in other papers, i think it's a problem with viral components or the titer. The problem is that when I use low dilutions it's ok but i have a 30% efficiency (and I want at least 70-80%) but when I raise concentration cells start to die.
By the way how do you measure viral titer?
VIral titers are typically measured accurately via FACS, if you have a fluorescent reporter on your construct. Our LV have GFP following either an IRES or 2A peptide sequence. That usually takes too much time & money, so I typically just titrate my virus in a dilution series (1:10, 1:100, 1:1000, 1:5000, etc.) on equal numbers of cells per well. Then I would pick the well that had the best expression after 2 days (the time it takes for the GFP to be synthesized, folded, and fluorescing). I choose the well w/ 70-80% infection of cells as my dilution, which is usually 1:5000.
Our packaging system involves: LV gene carrier, pRSV, pMDL, VSVg (the last 3 plasmids can be found on Addgene, I believe?).
Ok, we do the same things, maybe I have some problems during the production. Do you use polybrene on your infected cells?
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