The Tet-Off Tetracycline-regulated expression system
This Tetracycline-regulated expression system is comprised of two vectors: 
  • A regulatory vector (upper part of illustration)
  • An expression vector (lower part of illustration)
The regulatory vector encodes for the tranactivator protein (which is a fusion between Tet repressor DNA binding domain and transcriptional activation domain of VP16). The transactivator  can induce the expression of our gene of interest.
The expression vector contains our gene of interest under the regulation of a modified CMV promoter. This modified CMV promoter contains tet-op sites that can bind the transactivator protein, and enable transcription of our gene.
Following transfection of the two vectors into mammalian cells and addition of tetracycline (or doxycycline) to the growth medium, the tetracycline molecule binds the transactivator protein and prevents the transcription of our gene (right arrow).
Upon removal of the tetracycline from the medium, the transactivator protein will bind to the tet-op sequences in the CMV promoter, and expression of our gene of interest will commence (left arrow).


When establishing a Tet-off  system, the following steps should be performed:
  1. Transfect the regulatory vector into a mammalian cell line and select colonies resistant to G418.
  2. Choose the best clone by transiently transfecting each of several clones with an expression vector containing GFP reporter gene.
  3. Choose the best clone according to two parameters: A. high levels of GFP expression upon induction B. the best repression of GFP expression in the presence of tetracyclin.
  4. After choosing the best clone transfect it with the expression vector contaioning the BHR1 gene and create stable cell lines.
  5. Screen these cell lines for optimal expression of BHR1 protein and its suppression in the presence of tetracyclin.