Can you better summarize the content of your plasmid? Are both the ligand (rtTA) and tet-promoter-gene included in your plasmid? Is there any other constitutive promoter upstream of your target sequence?

Hi Christina Schäfer

Sometimes this 'leaky ' expression can occur. Perhaps there is some endogenous expression of your protein or the TetOn system has been corrupted somehow in certain cells. This can provide these cells with a selection advantage that makes them dominant and then your cells will all eventually be expressing the protein without doxycyline!

The way our lab solved this problem was to isolate single cells and culture them until we had colonies then test expression with and without doxycycline via Western Blot. We could then tell which clones were appropriate and did not leak protein expression. This does mean that your cell population will be homogenous but this is usually not a problem for protein production or conditioned media.

Hope this helps!

Hi Angelo Serani,

both the ligand (rTA) and the tet-responsive promotor as well as the gene of interest are in the same plasmid. I am using only one plasmid for the transduction. Upstream of my gene of interest is only the Tet-responsive promotor.

I also removed everything from the TetOn-system except the tet-responsive promotor, but the cells are still expressing the gene of interest.

Hi Jack Hopkins,

thank you for your reply. What do you exactly mean with corrupted?