Binary AdEasy vector systems designed for Tet-ON or Tet-OFF regulated control of transgene expression

Here, we describe the construction of a set of binary adenovirus vectors encoding for a tetracycline-regulatable expression cassette and the Tet-ON or the Tet-OFF transcriptional activator proteins from a single viral chromosome. The rabies virus glycoprotein was cloned into the E1 region and the tetracycline activator proteins were inserted in both orientation in place of the E3 region. To further restrict background transcription, we also introduced a Lac repressor protein based roadblock to transcription elongation. To make the system more versatile it has been engineered into the commonly used AdEasy system. We show that rabies virus glycoprotein expression is tightly regulated with an essentially undetectable basal expression and a several 100-fold induced expression. In our vector backbone, the Tet-ON and the Tet-OFF systems appears to work with essentially the same efficiency. Thus, the choice of principle can be based on whether a positive or negative regulation of reporter gene activity is desirable. Taken together our results suggest that the binary vectors described here should be a valuable addition to the repertoire of viral vectors used in basic and medical research.