Tight regulation of transgene expression by tetracycline-dependent activator and repressor in brain
Tight regulation of transgene expression by tetracycline-dependent activator and repressor in brain
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DOI：
10.1111/j.1601-183x.2005.00139.x
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发表时间：
2006-02-01
影响因子：
2.5
通讯作者：
Kida, S
中科院分区：
文献类型：
--
作者：
Uchida, S;Sakai, S;Kida, S
时间和空间调控基因突变的方法将为研究脑中基因功能提供强有力的策略。为了开发这些方法，我们已经建立了一个严格调控的系统，在前脑中使用四环素（Tc）依赖性转录激活因子（rtTA）和阻遏物（TetR-Kruppel相关框）的转基因表达。在该系统中，阻遏物在不存在多西环素（Dox）的情况下与Tc-应答元件（TRE）结合，导致对由rtTA与TRE的弱结合引起的TRE-mediated转录的泄漏激活的阻遏。在施用Dox后，仅激活剂结合TRE并激活转录。我们在培养细胞中测试了该系统，通过使用内部核糖体进入位点（IRES）双顺反子表达两种调节剂。在COS-1，HeLa和SHSY 5 Y细胞中，在Dox的存在下，由rtTA导致的泄漏转录激活被抑制，而不降低激活的转录水平。使用该系统，产生在α CaMKII启动子的控制下在前脑中使用IRES表达两种调节剂的转基因小鼠，并与携带TRE-dependent报告基因转基因的转基因小鼠一起繁殖。在成年双转基因小鼠前脑的逆转录-聚合酶链反应和原位杂交分析中，Dox处理诱导报告基因mRNA的表达，这在处理前和处理后撤销Dox后未检测到。这些结果表明，该系统允许在前脑中以Dox依赖的方式严格调节转基因表达，并将有助于研究大脑中的基因功能。
Methods to temporally and spatially regulate gene mutations will provide a powerful strategy to investigate gene function in the brain. To develop these methods, we have established a tightly regulated system for transgene expression in the forebrain using both a tetracycline (Tc)-dependent transcription activator (rtTA) and a repressor (TetR-Kruppel-associated box). In this system, the repressor binds to the Tc-responsive element (TRE) in the absence of doxycycline (Dox), leading to the repression of leaky activation of TRE-mediated transcription caused by weak binding of rtTA to TRE. Upon Dox administration, only the activator binds to TRE and activates transcription. We tested this system in cultured cells by bicistronically expressing both the regulators using an internal ribosome entry site (IRES). In COS-1, HeLa and SHSY5Y cells, leaky transcription activation led by rtTA in the absence of Dox was repressed without decreasing the level of activated transcription in the presence of Dox. Using this system, transgenic mice were produced that express both the regulators using IRES in the forebrain under the control of the alpha CaMKII promoter and were bred with transgenic mice carrying the TRE-dependent reporter transgene. In reverse transcription-polymerase chain reaction and in situ hybridization analyses of the forebrain in adult double transgenic mice, the treatment of Dox induces reporter mRNA expression, which was not detected before the treatment and after the withdraw of Dox following the treatment. These results indicate that this system allows the tight regulation of transgene expression in a Dox-dependent fashion in the forebrain and will be useful in investigating gene function in the brain.