Gene Targeting to Study Light-induced Circadian Changes

描述(申请人提供)：昼夜节律已被广泛研究 将其作为行为模型进行研究，因为它们具有高度的预测性 和在物种内的再生性，而且因为几种成分和 调节机制从果蝇到人类都是保守的。分子 对这一过程的剖析已经开始在小鼠身上使用基因敲除和转基因 战略。一般而言，在以下情况下，这些方法的信息可以最大化 基因的表达或切除可以限制在所需的组织中，并且 控制住了。这些方法已成功地用于研究 学习和记忆，但还没有被应用于调节 昼夜节律。造成这种情况的一个主要原因是，一个可靠的战略并没有 还没有开发出可以针对所需基因的特定表达来 视交叉上核(SCN)的视网膜突起细胞是一种 哺乳动物的昼夜节律调节器。 这里将开发一种有针对性的方法，以实现相对具体的目标 一种基因产物在SCN视网膜前神经元中的表达，即 昼夜节律时钟被重置的初级细胞 环境信号，如光。以显示目标定位的效用 策略中，Cre重组酶将在这些细胞中表达。组织特异性 Cre介导的基因切除将使用现有的记者进行演示 系统。一旦得到验证，表达Cre的小鼠将与现有的 诱骗NMDA1受体小鼠测试NMDA受体在体内的假设 视网膜感觉神经元是光诱导相移所必需的。 将来可能会采用相同的目标系统来研究假定的 其他信号蛋白参与光诱导的细胞重置 生物钟，如CAM激酶II、NOS、cGMP和cAMP依赖蛋白 蛋白激酶、贴图蛋白激酶和CREB。此外，相同的目标系统应该具有 在调查时钟的各个组件中的效用，例如PER、CRY、 BMAL和时钟蛋白。

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.