Inducible and reversible Clock gene expression in brain using the tTA system for the study of circadian behavior.
Inducible and reversible Clock gene expression in brain using the tTA system for the study of circadian behavior.
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DOI：
10.1371/journal.pgen.0030033
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发表时间：
2007-02-23
期刊：
影响因子：
4.5
通讯作者：
Takahashi, Joseph S.
中科院分区：
文献类型：
--
作者：
Hong, Hee-Kyung;Chong, Jason L.;Song, Weimin;Song, Eun Joo;Jyawook, Amira A.;Schook, Andrew C.;Ko, Caroline H.;Takahashi, Joseph S.
哺乳动物昼夜节律振荡的机制是细胞自主的，由一组形成转录自动调节反馈环的基因产生。虽然这些“时钟基因”在动物中是很保守的，但它们的具体功能仍有待充分了解，它们在中枢与外周昼夜节律振荡器中的作用仍有待确定。我们利用体内诱导型四环素控制的反式激活因子（tTA）系统，以组织特异性和时间控制的方式有条件地调节Clock基因的表达。通过使用分泌颗粒蛋白II驱动tTA表达，视交叉上核和脑定向表达tetO：：ClockΔ19显性负转基因延长了小鼠的昼夜运动节律周期，而过表达tetO：：Clockwt野生型转基因缩短了该周期。饮用水中低剂量（10 μg/ml）的强力霉素（Dox）有效地灭活了tTA蛋白，使tetO转基因沉默，并使昼夜节律周期恢复到野生型状态。重要的是，低剂量但不高剂量的Dox是完全可逆的，并导致tetO转基因的快速再激活。tTA调节的转基因表达的快速时间过程表明，CLOCK蛋白是脑中Dox依赖性诱导/抑制动力学的极好指标。有趣的是，该系统中昼夜节律周期的每日读数提供了体内tTA反式激活状态的实时读数。总之，tTA系统可以在中枢神经系统中以组织特异性、条件性和可逆的方式操纵生物钟基因表达。这里开发的具体方法应该具有普遍适用性的大脑和行为的研究在小鼠。虽然在揭示哺乳动物生物钟的分子机制方面取得了重大进展，但以前的工作主要集中在种系突变和体外分析方法上。为了解决时钟基因的功能，有必要开发工具来操纵生物钟基因在体内的条件和组织特异性的方式。我们报告这样一种方法使用四环素反式激活系统。尽管Bujard及其同事在10多年前在转基因小鼠中开发了“泰特”系统，但在中枢神经系统中成功使用泰特系统的例子仍然相对较少。Clock基因在小鼠视交叉上核和脑中的转基因表达调节昼夜运动节律的周期长度。饮水中低剂量的强力霉素可抑制上述作用。重要的是，低剂量但不高剂量的强力霉素是完全可逆的，并导致Clock转基因的快速重新激活。综上所述，四环素控制的反式激活因子系统可以在中枢神经系统中以组织特异性、条件性和可逆的方式操纵生物钟基因表达。这里开发的具体方法应该具有普遍适用性的大脑和行为的研究在小鼠。
The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these “clock genes” are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus– and brain-directed expression of a tetO::ClockΔ19 dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clockwt wild-type transgene shortened the period. Low doses (10 μg/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse. Although significant progress has been made in unraveling the molecular mechanism of circadian clocks in mammals, previous work has focused on germline mutations and in vitro methods for analysis. To address the function of clock genes, it is necessary to develop tools to manipulate circadian genes in a conditional and tissue-specific manner in vivo. We report such an approach using the tetracycline transactivator system. Despite the development of the “tet” system in transgenic mice over 10 y ago by Bujard and colleagues, there are still relatively few examples of the successful use of the tet system in the central nervous system. Transgenic expression of the Clock gene in the suprachiasmatic nucleus and brain of mice regulated the period length of circadian locomotor rhythms. These effects could be inhibited by low doses of doxycycline in the drinking water. Importantly, low, but not high, doses of doxycycline were completely reversible and led to a rapid reactivation of the Clock transgenes. In summary, the tetracycline-controlled transactivator system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.
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