I transfected a cell line with pRNAi vector and then added tetracyclin as an inducer of its promoter. How long do I have to wait if I want tetracyclin to induce the promoter of my plasmid and knock down my target gene?
Are you looking at mRNA or protein? For GFP fluorescence, the transfected RNAi vectors I use (not pRNAi, so this may not be comparable), there is little knockdown at 24 hr, some at 48 hr, and what is probably maximal knockdown at 72 hr (96 hr looks the same as 72 hr). I have not done the full time course for mRNA knockdown, but by 48 hr there is usually significant knockdown. Hope that helps.
I agree with Kathy, we have about the same time course (although it's a different setup, we are using cells stably transformed with an inducible lentiviral vector expressing an shRNA). We do our experiments after 3 days, by that time decrease of the level of our target mRNA is about maximal.That's true also for our protein, but that will depend on the turnover of the protein, ours is a rapid turnover protein.
As Jean-Paul already mentioned, the length of induction, and level of depletion will depend on the half-life of the protein that you deplete, and its amount in the cell. I had to wait for 4 days for about 90% depletion (evaluated by western-blot). This was true for using RNA oligoes or for expressing shRNA. Also, The concentration of Tetracyclin may influence the time required for depletion. If the inducible promoter wont be induced to its full potential (lower Tet concentration) it may require longer time to deplete the mRNA of the protein of interest.
Good luck!
Robert
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