A modified Tet-ON system minimizing leaky expression for cell-type specific gene induction in medaka fish
A modified Tet-ON system minimizing leaky expression for cell-type specific gene induction in medaka fish
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DOI：
10.1111/dgd.12743
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发表时间：
2021-08-26
影响因子：
2.5
通讯作者：
Miyaji, Mary
中科院分区：
文献类型：
--
作者：
Hosoya, Osamu;Chung, Myung;Miyaji, Mary
Tet-on系统是在时间和空间上控制可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系统来诱导转基因在青冈鱼的杆状光感受器中特异性地表达。我们的改进的反向四环素控制的转录反式激活因子(RtTAm)具有5个氨基酸取代，极大地改善了青冈鱼转基因的泄漏性。我们建立了一个转基因株系，携带由非洲爪哇视紫红质启动子驱动的rtTAm基因和四环素反应元件(Tre)，随后是绿色荧光蛋白(GFP)基因。我们证明了在多西环素存在的情况下(受精后9天)，GFP荧光仅限于鱼体的杆状感光器。GFP荧光强度随多西环素作用时间的延长而增强，最长可达72 h，且呈剂量依赖性(5~45µg/ml)。这些发现表明，使用rtTAm的Tet-on系统允许对转基因表达进行时空控制，至少在青冈鱼的杆状光感受器中是这样。
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 mu g/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.