I am using a Tet-On system to observe how over-expression of a certain gene affects cell proliferation. I have my cell line with my gene of interest and an EGFP control line. Using western blot I found that the minimum doxycycline conc. needed to get visible expression in my experimental line is about 0.25ul/ml and confirmed there is no expression in my control. I plated both cell lines and added the dox. Unfortunatly, I am seeing almost identical decreased proliferation rates in both my experimental line and control which isn’t optimal. I know doxycycline can be toxic to cells however I wasn’t expecting a conc. as low as 0.25ul/ml would have a significant effect on cell viability. Any ideas of why this might be happening? Am I using a bad assay design for these inducible systems?
Hi
First of all, 0.25ul/ml is not a concentration per se. It will be in ug/ml or mg/ml. Its possible you are using too much dox. I have used this system (0.9ug/ml) in the past with no problems. Further, be sure to change dox everyday.
Doxycyclin is an inhibitor of protein synthesis at ribosomal level. If your concentration is too high it may ecplain your problems.
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