I have previously treated a wild-type cell line with several drugs and found the effective drug concentrations that are toxic to cells. I then infected the same cell line with a virus (expressing shRNA) and selected with puromycin to generate a pool of stably infected cells. It now appears that even the control shRNA-infected cells are nearly resistant to the same panel of drugs. Does anyone have experience with viral infection making cells less sensitive to drug treatments? I am seeing this effect in the control (non-targeting shRNA) infected cells, so I am wondering if I should significantly increase my drug doses.
Hi Leila, as some changes has been occurred during generation of stable cell line to generate shRNA expressing virus. So your experiment on different panel of drugs should consider non targeting shRNA infected cells with virus vector as control to determine the toxic concentration of these drugs, and then apply these concentrations on stabilized cell expressed shRNA.
regards
Its the effect of puromycin coming in the vapor when the cells are within incubator. Puromycin is a powerful killer and hence, over time your uninfected cells maintained along with infected cells acquired resistance to puromycin and hence it shows drug resistance. You might find still puromycin killing these cells if you add puromycin in it, but would not kill completely.
You can overcome this by
1. Thawing fresh wild type cells which were frozen before your shRNA expedition.
2. Put the puromycin selection cells in a separate incubator.
Good Luck
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