Lost of Tet-on Vector - (Apr/30/2013 )

Hello, I have generated a tet-inducible model using TGFB. When I first made the cell lines it worked beautifully. Now it seems like the Tet-on vector is not regulating the expression of the TGFB. I made sure to use a cell line that had a low basal expression of TGFB and now the expression of TFGB is high in these cells before the Doxycycline is even added. Has anybody else had any problems with a Tet-on vector before? Im new to transfections and any help will be greatly appreciated!

FBS often contains tetracycline (doxycycline analogue) if it is from the EU, USA or Asia, check the source of your FBS and make sure it is tet-free. As far as I know, only New Zealand FBS is tet-free.

Thank you for the reply!

I am using a tet-free FBS from clontech, where I got the Tet-on Vector. I think it should be fine, it is the same FBS that I used right after transfection and it worked then, however, not it's not.

The tet-operon does also tend to be a bit leaky, though I don't think as much as you are seeing. Unfortunately I don't know of any way around this.