Establishment of a Tetracycline-Off System using a <i>piggyBac</i>-based Vector as a Gene Functional Analysis Tool for the Temporal Targeting of Gene Expression

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  • Establishment of a tetracycline-off system using a piggyBac-based vector as a gene functional analysis tool for the temporal targeting of gene expression

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To facilitate gene functional analysis systems in non-model insects, we focused on the Tetracycline-Off (Tet-Off) system, a binary expression system using a tetracycline controlled transactivator protein (tTA) and its binding site, the tetracycline operator sequences (tetO). The Tet-Off system can control gene expression temporally by adding or removing tetracycline (Tc) or doxycycline (Dox), which inhibits the transactivational activity of tTA. In this study, we established a Tet-Off system applicable to a wide variety of insects by constructing the Drosophila heat shock protein 70 (hsp70)-tTA and tetO-Enhanced Green Fluorescent Protein (EGFP) vectors using piggyBac, the most widely used transformation vectors in insects to date. The effectiveness of these vectors was examined using transgenic flies. We show that (1) EGFP fluorescence was efficiently induced by the hsp70 promoter using the Tet-Off system, (2) Tc or Dox can effectively inactivate tTA to repress gene expression under tetO in a concentration dependent manner, and (3) removal of Tc or Dox from the diet can recover the transactivational activity of tTA, also in a concentration- and time-dependent manner. Since Drosophila hsp70 promoter and piggyBac are useful in many insects, our results indicate that the Tet-Off system used in this study provides an ideal opportunity for examining the function of genes in non-model insects as well as in Drosophila.<br>

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