Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish
Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish
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DOI：
10.1073/pnas.1007799107
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发表时间：
2010-11-16
影响因子：
11.1
通讯作者：
Weidinger, Gilbert
中科院分区：
文献类型：
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作者：
Knopf, Franziska;Schnabel, Kristin;Weidinger, Gilbert
基因表达的时空调控系统对于发育研究是必不可少的，对于成年模式生物的研究尤其重要。我们提出了两个修改后的双重诱导TetON系统的组织特异性条件控制的基因表达在斑马鱼的基础上（i）四环素诱导的转录激活因子（TetActivator）融合的配体结合结构域的突变糖皮质激素受体（TetA-GBD）和（ii）TetActivator融合的结构域的蜕皮激素受体（TetA-EcR）。这两个系统显示出强烈的诱导四环素响应启动子后，适当的配体（多西环素和地塞米松TetA-GBD，和多西环素和tebufenozide TetA-EcR），和不可检测的泄漏相比，经典TetActivators。在心脏或CNS中特异性表达TetA-GBD的转基因系与不同Tet-responsive转基因系的组合允许胚胎和成人中基因表达的条件性和组织特异性控制。重要的是，诱导是完全可逆的，并可通过所用药物的剂量进行调节。TetA-EcR系统避免了地塞米松可能的副作用，并在斑马鱼和哺乳动物细胞中显示出改善的灵敏度。这些结果表明，双重诱导TetON系统是斑马鱼中可逆和非常严格控制的条件基因表达的方便工具。
Systems for spatial and temporal control of gene expression are essential for developmental studies and are of particular importance for research in adult model organisms. We present two modified dually inducible TetON systems for tissue-specific conditional control of gene expression in zebrafish based on (i) a tetracycline inducible transcriptional activator (TetActivator) fused to the ligand binding domain of a mutated glucocorticoid receptor (TetA-GBD) and (ii) a TetActivator fused with a domain of the Ecdysone receptor (TetA-EcR). Both systems showed strong induction of tetracycline-responsive promoters upon administration of the appropriate ligands (doxycycline and dexamethasone for TetA-GBD, and doxycycline and tebufenozide for TetA-EcR), and undetectable leakiness when compared with classical TetActivators. Combinations of transgenic lines expressing TetA-GBD specifically in the heart or the CNS with different Tet-responsive transgenic lines allows conditional and tissue-specific control of gene expression in embryos and adults. Importantly, induction is fully reversible and tunable by the doses of drugs used. The TetA-EcR system avoids the possible side effects of dexamethasone and displays improved sensitivity both in zebrafish and in mammalian cells. These results show that dually inducible TetON systems are convenient tools for reversible and very tightly controlled conditional gene expression in zebrafish.