Has anyone succeeded in transduction the RAW or BMDM cells with lentivirus (PLKO.1) without puromycin selection? The BMDM selection could be a little bit difficult if I need large scale of cells for sequencing analysis for example, I suppose if I increase the dosage of the virus I will manage to get nice knocking down without puromycin selection. Does anyone have experience on that..
I have provided lentivirus to infect RAW macrophages and it worked very well, but we did need to use a very high titre virus. We used a CMV-GFP which normally produces virus very very well (1e10), so you may just need to apply more virus. A word of caution: polybrene adversely affected these cells. We had to use Retronectin instead.
Thanks for your answer Jill. When you say polybrene adversely affected the RAW cells, do you mean they reduce the transduction effecacy? I used 8ug/ml polybrene overnight during the lentivirus infection, the cell looked healthy though... Do you think polybrene might also not be good for BMDM lentivirus infection as well?
No, polybrene should increase your transduction efficiency but in some cells the polybrene alters their characteristics. In mesenchymal stem cells, polybrene causes the cells to stop proliferating. I think for the RAW cells they either stopped proliferating or stopped their migration-like behaviour. I'll check with the student who was actually culturing the cells.
OK thanks. In my situation, the cells looked fine under microscope, although I did not quantify the proliferation by assays... Have you tried the BMDM cells too?
No, only the macrophages. It's the kind of thing you have to assess on an individual basis - some cells seem completely unaffected by polybrene and others react badly.
Jill Muhling Did u expressed shRNA or full length gene by transduction ?
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