Tetracycline-regulated expression implemented through transcriptional activation combined with proximal and distal repression.

Tetracycline-regulated expression systems are widely used to control ectopic gene expression in mammalian cells. However, background or "leaky" expression in the "off" state can limit applications that require control of expression at low levels. In this work we have engineered a tetracycline-regulated expression system with an improved range of control and lower background expression. To lower background expression without diminishing the controllable expression range, we designed a feed-forward scheme that repressed both expression of the gene of interest and the transcriptional activator. By using a tetracycline-responsive repressor that can modify chromatin and repress transcription over short and long distances, we were able to repress these two expression targets using a single tetracycline-responsive genetic element. This dual-targeting repressor/activation system demonstrated decreased background expression in its "off" state and a 25-fold range of expression in response to doxycycline. This study demonstrates that genetic circuits can be improved by leveraging trans-acting factors with long-range capabilities.