TRANSGENIC MOUSE REPORTERS OF CIRCADIAN GENE EXPRESSION

昼夜节律基因表达的转基因小鼠报告基因

• 批准号: 6330345
• 负责人: DOUGLAS G MCMAHON
• 金额: $ 7.25万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6330345
• 关键词: animal tissue; bioimaging /biomedical imaging; biological clocks; brain imaging /visualization /scanning; circadian rhythms; fluorescent dye /probe; forskolin; fos protein; gene induction /repression; genetic promoter element; genetic strain; genetic transcription; genetically modified animals; glutamates; green fluorescent proteins; laboratory mouse; neurophysiology; reporter genes; suprachiasmatic nucleus; visual stimulus

The physiology and behavior of virtually all eukaryotes, including humans, exhibits a pervasive daily rhythmicity. Many critical daily rhythms are endogenous, driven by an internal master pacemaker or biological clock, with a free-running period of about a day (i.e., circadian). The suprachiasmatic nuclei (SCN) of the hypothalamus are the apparent site of the master biological clock in mammals. Recent research has revealed that (i) the ability to generate neurophysiological circadian rhythms reside in single SCN neurons, and (ii) the fundamental mechanisms for rhythms resides in single SCN neurons, and (ii) the fundamental mechanisms for rhythms generation within these neurons involve transcriptional negative feedback loops among putative circadian clock genes. A critical for circadian neurobiology is to establish means to monitor real time circadian gene expression in single SCN neurons so that the precise roles of putative clock genes, and the linkage of the intracellular molecular clock with its neurophysiological output, can be investigated in detail. Toward these ends we have created three kinds three of transgenic mice incorporating the reporter constructs fosdGFP. Toward these ends we have created three kinds of transgenic mice incorporating the reporter constructs fosdGFP, (dGFP) appropriate for assaying dynamic gene expression. Our hypothesis is that SCN neurons from fosdGFP and perdGFP mice will display circadian gating of induction and circadian rhythms in dGFP fluorescence correlating with the previously documented endogenous rhythms in c-fos and mper1 RNAs. We propose three specific aims to test and expand our hypothesis. Aim 1-Intact Animal Induction Experiments-to validate the function of the transgene reporters. Aim 2 In Vitro Induction Experiments-to test for acute induction of transgene reporters. Aim 2- In Vitro Induction Experiments-to test for acute induction of transgene reporters in vitro. Aim 3-In Vitro Circadian Experiments-to test for circadian rhythms in fos or per-driven gene expression. Successful completion of these aims will aims will establish promoter driven dGFP transgene reporters as an approach for assessing the molecular circadian rhythms in individual SCN neurons. This will provide a methodological foundation for expanded future research on the molecular basis of the mammalian SCN clock which should increase our understanding of the mechanism of seasonal affective disorder, age-related disruption of biological timing, and jet lag.

几乎所有真核生物（包括人类）的生理和行为表现出普遍存在的每日节奏性。许多关键的每日节奏是内源性的，由内部的起搏器或生物时钟驱动，自由运行时间约为一天（即昼夜节律）。下丘脑的上核（SCN）是哺乳动物主要生物钟的明显位点。最近的研究表明，（i）产生神经生理昼夜节律的能力位于单个SCN神经元中，以及（ii）节奏的基本机制位于单个SCN神经元中，以及（ii）这些神经元中的基本机制参与这些神经元的基本机制。推定的昼夜节律钟基因中的转录负反馈回路。昼夜节律神经生物学的关键是建立单个SCN神经元中的实时昼夜节律基因表达的方法，以便可以详细研究推定时钟基因的精确作用，并与细胞内分子时钟的联系以及细胞内分子时钟的联系。在这些目的方面，我们创建了三种结合记者构造FOSDGFP的转基因小鼠的三种。在这些目的方面，我们创建了三种结合报告基因构建的转基因小鼠，即FOSDGFP（DGFP），适合分析动态基因表达。我们的假设是，来自FOSDGFP和PERDGFP小鼠的SCN神经元将在DGFP荧光中显示与先前记录的C-FOS和MPER1 RNA中的内源性节律相关的循环和昼夜节律节奏。 我们提出了三个特定的目的，以检验和扩展我们的假设。 AIM 1-INTACT动物诱导实验验证了转基因报告者的功能。 AIM 2体外诱导实验对转基因记者的急性诱导测试。 AIM 2-体外诱导实验对测试，以便在体外急性诱导转基因记者。 AIM 3-IN体外昼夜节律实验对FOS或驱动基因表达中的昼夜节律测试。这些目标的成功完成将建立启动子驱动的DGFP转基因记者，以评估单个SCN神经元中的分子昼夜节律节奏。这将为哺乳动物SCN时钟的分子基础扩展未来的研究提供方法论基础，这应该提高我们对季节性情感障碍，与年龄相关的生物时机的破坏和喷气滞后的理解。