Gene Targeting to Study Light-induced Circadian Changes

描述（由申请人提供）：昼夜节律已被广泛 作为行为模型进行研究，因为它们具有高度的可预测性， 和再现性，因为几个组件和 调节机制从果蝇到人类都是保守的。分子 已经开始使用基因敲除和转基因技术在小鼠中剖析这一过程。 战略布局一般来说，如果满足以下条件，可以最大限度地利用这些方法提供的信息： 基因的表达或切除可限于所需组织， 控制。这些方法已成功地用于调查 学习和记忆，但尚未应用于调节 昼夜节律一个主要的原因是，一个可靠的战略， 已经开发出可以特异性靶向所需基因的表达， 视交叉上核（SCN）的视网膜受体细胞， 哺乳动物的昼夜节律调节器。 这里将制定一种目标确定方法， SCN的视网膜受体神经元中基因产物的表达，即 其中生物钟响应于 环境信号，如光。为了展示目标的效用 策略，CRE重组酶将在这些细胞中表达。组织特异 CRE介导的基因切除将使用已建立的报告基因进行证明。 系统一旦得到验证，表达CRE的小鼠将与现有的 foxed NMDA1受体小鼠，以检验NMDA受体在 视网膜受体神经元是光诱导的相移所必需的。 同样的靶向系统可能会在未来适用于研究推定的 参与其他信号蛋白在光诱导的复位的 生物钟，如CAM激酶II、NOS、cGMP和cAMP依赖性蛋白 激酶、MAP激酶和CREB。此外，同样的目标系统应该 在研究时钟的各个组件时的实用程序，例如PER，CRY， BMAL和CLOCK蛋白。

DESCRIPTION (provided by applicant): Circadian rhythms have been extensively studied as a behavioral model because of their high degree of predictability and reproducibility within a species, and because several components and regulatory mechanisms are conserved from Drosophila to humans. Molecular dissection of this process has begun in mice using knockout and transgenic strategies. In general, information from these approaches can be maximized if expression or excision of a gene can be restricted to a desired tissue and controlled. These approaches have been successfully used to investigate learning and memory, but have not yet been applied to the regulation of circadian rhythms. A major reason for this is that a reliable strategy has not yet been developed that can specifically target expression of a desired gene to the retinorecipient cells of the suprachiasmatic nucleus (SCN), a primary circadian regulator in mammals. A targeting approach will be developed here to achieve relatively specific expression of a gene product in the retinorecipient neurons of SCN, i.e. the primary cells in which the circadian clock is reset in response to environmental signals such as light. To show the utility of the targeting strategy, CRE recombinase will be expressed in these cells. Tissue-specific CRE-mediated gene excision will be demonstrated using an established reporter system. Once validated, the CRE-expressing mice will be breed with existing foxed NMDA1 receptor mice to test the hypothesis that NMDA receptors in retinorecipient neurons are critically required for light-induced phase shifts. The same targeting system may be adapted in the future to study the putative involvement of other signaling proteins in light-induced resetting of the circadian clock, such as CAM kinase II, NOS, cGMP- and cAMP-dependent protein kinases, MAP kinases and CREB. Moreover, the same targeting system should have utility in investigating individual components of the clock, such as PER, CRY, BMAL and CLOCK proteins.