I'm looking to transduce primary microglia with lentivirus carrying an shRNA vector that has a puromycin selection component on the vector. However, I see that the methods of some papers where they use lentivirus and primary microglia don't specify anything about selection of cultures by addition of antibiotic, perhaps because primary microglia don't proliferate once isolated and plated from the mixed glial culture. The papers where I do see use of antibiotic to select against non-transduced cells are where they use a stable cell line (i.e. BV-2).
So the question is, to select with puromycin or not to select with puromycin? I suppose I could always control for knockdown by Western / RT-PCR. Any help would be greatly appreciated!
Dear Edward Koellhoffer,
I think it will depend on the specific experimental purpose. Normally, lentivirus infection without selection will be enough for most of the experiments. However, if you want to get a relatively pure infected population, then you can do the selection. Another thing about infecting microglia by lentivirus is that the infection process will significantly acivate micrgola. That means the expression levels of many inflammatory factors will significantly increased in microglia after the infection. So, if you want to do some experiments related to inflammatory response of microglia, i don't think infecting them with lentivirus is a perferct model system.
I don't know much about microglia but I wouldn't think primary cells would replicate enough for drug selection to really work. For shRNA studies you do need a pretty high transduction efficiency, I know that LV works very well in C6 glioma cells, not sure if that's relevant to you or not.
Thanks Jill and Yingjun for your responses! Jill, I kind of agree that the primary cells won't replicate for selection to really work. With primary microglia, you culture them in a mixed culture with astrocytes, and the astrocytes produce M-CSF which causes the microglia to proliferate and grow. However, once you isolate the microglia off the astrocytes, there's no more M-CSF and so they don't proliferate. They really just sit on the plate waiting for you to do something to them. Which is actually another reason to use lentivirus because they're non-dividing at that point. That's probably why I don't see people using puromycin selection in their primary microglia work with lentivirus.
Ah OK, well if they're not proliferating then it sounds like drug selection definitely won't work. If you're getting good transduction efficiency then it doesn't really matter. If you're getting poor transduction efficiency you could always try AAV.
Be aware that microglia and microglial cell lines are difficult to transduce with lentivirus, at least with VSVG pseudotyping. I haven't tried other types of viruses.
These cells are also difficult to transfect, but after testing a number of reagent, I found that MirusTransIT-2020 is by far the best reagent to transfect BV-2 cells.
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