novel fusion protein that functions as an enhanced green fluorescent protein reporter and a tetracycline-controlled transcriptional activator

Binary expression systems are widely employed to analyze gene function in vivo using transgenic organisms. The tetracycline-off (Tet-Off) system, which is a binary expression system that uses a tetracycline-controlled transactivator protein (tTA) and its tetracycline operator sequence (tetO) binding site, was developed as a method for temporally controlling gene expression. To facilitate the use of the Tet-Off system in animal species other than the model organisms that are widely used for genetic analysis, we constructed two different fusion proteins containing enhanced green fluorescent protein (EGFP) as the reporter gene and tTA as the transactivator, in different configurations. We assessed the utility of these fusion proteins designated as tTA-EGFP and EGFP-tTA in transgenic fruit flies. We showed that, although EGFP of both fusion proteins was efficiently fluoresced, transcriptional activation occurred only by the tTA-EGFP fusion protein. Furthermore, tetracycline (Tc) and doxycycline (Dox) both effectively inactivated tTA-EGFP, repressing gene expression under tetO control in a concentration-dependent manner. Additionally, the removal of Tc or Dox from the diet can recover the transactivator activity of tTA-EGFP in a concentration- and time-dependent manner. The tTA-EGFP fusion protein will therefore be useful in the analysis of gene function in a wide range of animal species.