Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats
Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats
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DOI：
10.1073/pnas.0806213105
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发表时间：
2008-11-25
影响因子：
11.1
通讯作者：
Reichardt, Holger M.
中科院分区：
文献类型：
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作者：
Herold, Marco J.;van den Brandt, Jens;Reichardt, Holger M.
目前，在大鼠中产生功能丧失突变的工具有限。因此，我们已经开发了一个慢病毒单载体系统的普遍存在的shRNA表达的时间控制。在这里，我们报告转基因大鼠携带胰岛素受体特异性的shRNA转录从一个可调控的启动子，并确定伴随EGFP表达。在没有诱导剂强力霉素（Dox）的情况下，我们没有观察到siRNA表达。然而，在非常低的浓度下的Dox处理导致siRNA的快速诱导和INSR蛋白表达的消除。正如预期的那样，血糖水平升高，而胰岛素信号传导和葡萄糖调节受损。重要的是，这种表型是可逆的（即，停止Dox治疗导致INSR再表达和糖尿病症状缓解）。慢病毒系统提供了一个简单的工具，可逆的基因消融在大鼠和可用于其他物种，不能通过传统的重组技术进行操作。
Currently, tools to generate loss-of-function mutations in rats are limited. Therefore, we have developed a lentiviral single-vector system for the temporal control of ubiquitous shRNA expression. Here, we report transgenic rats carrying an insulin receptor-specific shRNA transcribed from a regulatable promoter and identified by concomitant EGFP expression. In the absence of the inducer doxycycline (Dox), we observed no siRNA expression. However, Dox treatment at very low concentrations led to a rapid induction of the siRNA and ablation of INSR protein expression. As anticipated, blood glucose levels increased, whereas insulin signaling and glucose regulation were impaired. Importantly, this phenotype was reversible (i.e., discontinuation of Dox treatment led to INSR re-expression and remission of diabetes symptoms). The lentiviral system offers a simple tool for reversible gene ablation in the rat and can be used for other species that cannot be manipulated by conventional recombination techniques.