Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.
Dev Growth Differ. 2021 Oct;63(8):397-405. doi: 10.1111/dgd.12743. Epub 2021 Aug 26.
The Tet-ON system is an important molecular tool for temporally and spatially-controlled inducible gene expression. Here, we developed a Tet-ON system to induce transgene expression specifically in the rod photoreceptors of medaka fish. Our modified reverse tetracycline-controlled transcriptional transactivator (rtTAm) with 5 amino acid substitutions dramatically improved the leakiness of the transgene in medaka fish. We generated a transgenic line carrying a self-reporting vector with the rtTAm gene driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) followed by the green fluorescent protein (GFP) gene. We demonstrated that GFP fluorescence was restricted to the rod photoreceptors in the presence of doxycycline in larval fish (9 days post-fertilization). The GFP fluorescence intensity was enhanced with longer durations of doxycycline treatment up to 72 h and in a dose-dependent manner (5-45 μg/ml). These findings demonstrate that the Tet-ON system using rtTAm allows for spatiotemporal control of transgene expression, at least in the rod photoreceptors, in medaka fish.
Tet-ON 系统是一种用于时空控制诱导基因表达的重要分子工具。在这里，我们开发了一种 Tet-ON 系统，以特异性诱导斑马鱼杆状光感受器中转基因的表达。我们对经过 5 个氨基酸替换的改良型反向四环素调控转录激活子（rtTAm）进行了修饰，从而显著降低了该转基因在斑马鱼中的本底表达。我们构建了一个携带自我报告载体的转基因系，该载体由非洲爪蟾视紫红质启动子和四环素反应元件（TRE）驱动，其后紧跟绿色荧光蛋白（GFP）基因。我们证实，在幼虫（受精后 9 天）存在强力霉素时，GFP 荧光仅限于杆状光感受器。随着强力霉素处理时间的延长（长达 72 小时）和剂量的增加（5-45μg/ml），GFP 荧光强度增强。这些发现表明，使用 rtTAm 的 Tet-ON 系统至少可以在斑马鱼的杆状光感受器中实现时空控制的转基因表达。
