We're using a working concentration of 1ug/mL Dox (every 48hr) for induction of protein expression with a Tet-on system. However, even after 96 hours we don't see expression of our GFP-tagged protein construct. This is a lenti-virus stably transfected HEK-293T cell line. The cells otherwise seem healthy and continue to be puromycin resistant.
Dear Elias J Fernandez
DOX 1ug/mL is a commonly used concentration, and higher concentrations may cause cytotoxicity.
I would question the sequence of the GFPtagged protein gene or suspect that the expression level of rtTA is low.
Best,
Narumi
Dear Elias J Fernandez,
I agree with above suggestion from Narumi Uno. HEK293T cell treated with Dox at 10ug/ml is fine in my experiment. Cell toxicity was usually oberserved at concentration of Dox higher than 50ug/ml. Target protein could be effeciently induced from a simialr TRE3G-based Tet-on system at 1ug/ml of Dox. So please sequence your GFP expression frame as well as rTA from your established cell line to ensure whether the GFP or transactivator could be expressed or not.
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