Genetic Analysis of Circadian Oscillator Hierarchy

描述(申请人提供)：昼夜节律存在于整个动物界的物种中，昼夜节律的紊乱会导致基于昼夜节律的睡眠障碍、轮班工人的适应失调和时差反应，并可能导致包括抑郁和季节性情感障碍在内的神经精神障碍。转录-翻译反馈环是生物钟机制的中心。时钟基因显性负突变的小鼠或BMal1基因零突变的小鼠的表型表明，这些转录因子对生物钟功能是必不可少的。在这个方案中，我们将产生时钟基因有条件干扰的小鼠，在这种情况下时钟基因可以以组织特异性的方式被破坏。这项工作将使用分子、生化和行为方法来定义时钟基因在核心转录机制中的作用。此外，组织特异性基因打靶策略将首次使我们能够解决与外周组织中振荡器的功能重要性有关的重要问题。我们的假设是时钟是一种不可替代的转录激活剂，是分子和生理节律所必需的。我们预计，大脑中的行为和分子节律将在大脑特有的时钟基因中断后被扰乱。在具有肝脏特异性时钟基因干扰的小鼠中，我们预计基于大脑的节律性将持续存在，但肝脏的局部节律性将被扰乱。这些小鼠将被用来评估这样的假设，即肝脏固有的节律性负责药物和毒素的节律性新陈代谢。这项拟议的研究将验证一种新的方法来扰乱特定组织的昼夜节律功能，使我们能够确定外周振荡器的生理意义。这些研究将作为建立昼夜节律振荡器等级的遗传解剖和特定组织中振荡器功能评估的“原则证明”。

DESCRIPTION (provided by applicant): Circadian rhythms are present in species throughout the animal kingdom, and disorders of circadian timing contribute to circadian-based sleep disorders, maladjustment of shift workers and during jet lag, and may contribute to neuropsychiatric disorders including depression and seasonal affective disorder. A transcriptional-translational feedback loop is at the center of the circadian clock mechanism. The phenotype of mice with a dominant-negative mutation in the Clock gene, or of mice with a null mutation in the Bmal1 gene, suggest that these transcription factors are essential for circadian clock function. In this proposal, we will generate mice with conditional disruption of the Clock gene, in which the Clock gene can be disrupted in a tissue specific manner. The work will use molecular, biochemical and behavioral approaches to define the contribution of the Clock gene in the core transcriptional machinery. Furthermore, the tissue-specific gene targeting strategy will for the first time allow us to address important issues regarding the functional importance of oscillators in peripheral tissues. Our hypothesis is that CLOCK is an irreplaceable transcriptional activator, necessary for molecular and physiological rhythmicity. We expect that behavioral and molecular rhythms in brain will be disrupted following a brain-specific disruption of the Clock gene. In mice with a liver-specific disruption of the Clock gene, we expect that brain-based rhythmicity will persist, but local rhythmicity in the liver will be disrupted. These mice will be used to assess the hypothesis that rhythmicity intrinsic to the liver is responsible for rhythmic metabolism of drugs and toxins. The proposed studies will validate a novel method for disruption of circadian function in specific tissues, allowing us to determine the physiological significance of peripheral oscillators. These studies will serve as a "proof of principle" establishing methods for genetic dissection of the circadian oscillator hierarchy and assessment of oscillator function in specific tissues.