Quantitative Trait Genes for Circadian Phenotypes

昼夜节律表型的数量性状基因

• 批准号: 8391544
• 负责人: John R Hofstetter
• 金额: --
• 依托单位: RLR VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391544
• 关键词: Address; Affect; Anterior; Area; Arousal; Behavior; Behavioral; Biochemical Pathway; Biological Assay; Biotechnology; Bipolar Disorder; Birds; Brain; Breeding; Candidate Disease Gene; Cell Culture System; Cell Nucleus; Cells; Characteristics; Chromosomes; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 12; Circadian Rhythms; Code; Communities; Congenic Mice; Controlled Study; Core Facility; Culture Media; Cultured Cells; Distal; Distant; Dose; Elements; Frequencies; Funding; Gene Dosage; Gene Targeting; Genes; Genetic; Genetic Recombination; Genome; Genomic Segment; Genomics; Goals; Harvest; Heritability; Human; Inbred Mouse; Inbred Strains Mice; Indiana; Jet Lag Syndrome; Laboratories; Light; Measures; Medical; Medical center; Mental Depression; Mental disorders; Modeling; Motor Activity; Mouse Strains; Mus; Outcome; Pathway Analysis; Patients; Pattern; Pennsylvania; Pharmaceutical Preparations; Phenotype; Polymerase Chain Reaction; Post-Traumatic Stress Disorders; Proteins; Quality of life; Quantitative Trait Loci; Regulatory Element; Reporting; Research; Resources; Rest; Role; Schizophrenia; Sleep; Sleep Disorders; Slice; Small Interfering RNA; Sorting - Cell Movement; Steroids; Strigiformes; System; Testing; Time; Transcript; Universities; Variant; Veterans; Work; behavior change; behavior influence; cellular engineering; circadian pacemaker; gene function; innovation; interest; mouse genome; mouse model; programs; research study; shift work; tool; trait

The proposed research will characterize two genomic regions of the mouse: one on Chromosome 1 and the other on 12. The region on 1 is not only important to many behaviors, but contains regulatory elements for hundreds of genes on distant chromosomes as well. The region on 12 has genes known to interact with components of the biochemical pathway called the circadian clock. This program has focused on differences in daily (circadian) timekeeping in strains of mice. Some strains wake up earlier each day while the others wake up later each day. The first is a model for humans who are "early birds" and the other a model for "night owls". The goal is identifying genes that underlie this difference. With former funding, intercrossed mouse strains produced new strains whose genomes carried elements influencing some heritable parts of daily timekeeping. The new elements came from another strain of "early birds." Importantly, the new elements imparted some of the characteristic to the new strains. The goal is pinpointing genes within the elements (small genomic segments) that impact timekeeping. The way forward is to associate changing behaviors with changing gene function. Measuring function requires three technically challenging assays: the quantitative real-time polymerase chain reaction (qRT-PCR); the microarray system; and a specialized cell culture system called gene dosage network analysis (GDNA). Hypothesis: Aspects of circadian timekeeping and sleep come from certain genes on mouse chromosomes 1 and 12. A comparison throughout a day of the use of a gene from one strain with its use in a strain whose behavior varies, tests the hypothesis. For chromosome 1 the comparison requires a microarray assay. This powerful assay simultaneously compares all functioning genes in one experiment. The material assayed is extracts of suprachismatic nucleus (SCN) of the anterior hypothanamus dissected from brain slices and harvested around the clock. A gene's use should reflect the behavior variations: activity or rest. If as a gene's usage spikes behavior also changes, then some of the activated genes must be behavior modulators. A comparison of an immediate gene-product from chromosomes 12 between the special lines of mice with qRT-PCR tests this hypothesis also. Again it is important to compare gene-product levels around the clock. If the function of the target gene (Zfp277) changes with the behavior, it is a clue that Zfp277 influences behavior. GDNA is for the most important candidate genes. The assay comprises special cultured cells engineered to light up as their circadian clocks are active. Furthermore, when the culture is challenged with a dose of steroid, then in synchrony once a day for six days, the entire culture lights up. Add a small interfering RNA (siRNA) corresponding to the complimentary strand of the candidate-gene's transcript to the culture's media, and the cells' ability to make the coded protein slows and stops. Should the missing protein interact with the cells' clockwork, not only flashing frequency but also levels of key clock genes (qRT-PCR assay) change. A candidate gene's part in a well-regulated sleep and arousal system follows from a positive assay. Fulfilling the proposal's goal also means continuing to breed new strains. Introducing and fixing the outcome of recombination generates not only informative but analysis-friendly genomic substrates. Since new strains developed go out to other laboratories, they serve the research objectives of others interested in this important segment of chromosome 1 as well as ours. This work capitalizes not only on the unique and irreplaceable mouse lines developed at the Indianapolis VA Medical Center but also the rich resources available in the Laboratory of Dr. Hogenesch at University of Pennsylvania and in the Medical Genomics and Biotechnology Core Facilities at Indiana University.

拟议的研究将表征小鼠的两个基因组区域：一个在染色体上和一个基因组区域。 其他12。1上的区域不仅对许多行为很重要，而且包含调节元素 遥远染色体上的数百个基因。 12个区域的基因已知可以与 生化途径的组成部分称为昼夜节律。 该计划的重点是小鼠菌株的日常（昼夜节律）计时差异。一些菌株醒来 每天早些时候，其他人每天晚些时候醒来。第一个是人类的模型 鸟类”，另一个是“夜猫子”的模型。目标是确定构成这种差异的基因。 借助以前的资助，间交叉的小鼠菌株产生了新的菌株，其基因组带有元素 影响日常计时的某些部分。新元素来自另一种早期的压力 鸟类。“重要的是，新元素赋予了新菌株的一些特征。 目的是指出影响计时的元素（小基因组段）中的基因。道路 向前是将不断变化的行为与不断变化的基因功能相关联。测量功能需要三个 在技​​术上具有挑战性的测定：定量实时聚合酶链反应（QRT-PCR）；微阵列 系统;和一种称为基因剂量网络分析（GDNA）的专门细胞培养系统。 假设：昼夜节律的各个方面来自小鼠染色体上的某些基因1 和12。在整天使用一个菌株的基因的一天中的比较与其在菌株中的使用 行为各不相同，检验假设。对于染色体1，比较需要微阵列测定。这 强大的测定同时比较一个实验中的所有功能基因。材料测定是 从脑切片和 全天候收获。基因的使用应反映行为的变化：活动或休息。如果作为基因 使用峰值行为也会改变，然后一些激活的基因必须是行为调节剂。 特殊小鼠的染色体12的直接基因产物的比较 QRT-PCR也检验了这一点。同样，重要的是要全面比较基因产物水平。如果 靶基因（ZFP277）的功能随行为而变化，这是ZFP277影响行为的线索。 GDNA用于最重要的候选基因。该测定包括设计为特殊培养的细胞 照亮他们的昼夜节律很活跃。此外，当培养物受到一剂类固醇的挑战时， 然后，每天一次同步六天，整个文化亮起了。添加一个小的干扰RNA（siRNA） 对应于候选人 - 基因到文化媒体的转录的免费链，以及 细胞使编码蛋白的能力减慢和停止。丢失的蛋白应该与细胞的相互作用 发条，不仅闪烁的频率，而且还会更改关键时钟基因（QRT-PCR测定）的水平。一个 候选基因在良好调节的睡眠和唤醒系统中的作用是从积极的测定中进行的。 实现提案的目标也意味着继续繁殖新的菌株。介绍和解决结果 重组不仅会产生信息性的，而且会产生分析友好的基因组底物。由于新菌株 开发向其他实验室，他们为对此重要的人感兴趣的其他人的研究目标服务 染色体1和我们的段。 这项工作不仅利用印第安纳波利斯VA开发的独特且不可替代的鼠标线 医疗中心，也可以在大学的Hogenesch博士实验室提供丰富的资源 宾夕法尼亚州以及印第安纳大学的医学基因组学和生物技术核心设施。