Project 5: High-Throughput Analysis of Gene Regulation

项目5：基因调控高通量分析

• 批准号: 6850577
• 负责人: William E BUNNEY
• 金额: $ 41.22万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850577
• 关键词: behavioral /social science research tag; bipolar depression; complementary DNA; gene expression; genetic regulation; genotype; high throughput technology; human tissue; major depression; messenger RNA; method development; neurogenetics; neuropathology; nucleic acid sequence; phenotype; polymerase chain reaction; psychobiology; serial analysis of gene expression

The purpose of this project is to identify gene expression abnormalities in specific brain regions that may underlie the maintenance and manifestation of mood disorders. The underlying hypothesis is that the altered biological states that accompany the behavior or emotional phenotypes are likely to be initiated, sustained, or modified by recognizable changes in gene expression patterns in specific brain regions, thus a simultaneous monitoring of tens of thousands of genes in multiple, inter-connected brain regions provides an excellent opportunity to define the neurological basis of mental disorders. The emphasis in this application is to develop and apply alternative high-throughput methodologies to measure gene expression, so as to overcome limitations of oligonucleotide microarrays. In Specific Aim 1, quantitative RT-PCR will be used to achieve greater flexibility and less dependence on current knowledge of gene function, to provide greatly improved specificity, sensitivity, and accuracy, thus allowing routine, in-depth validation of preliminary findings from other technological platforms or candidate genes from gene-specific, mechanistic hypotheses. In Aim 2, Serial Analysis of Gene Expression will be used to ensure comprehensiveness and to provide the opportunity to discover genes with hitherto unknown roles in mental disorders. In Aim 3, spotted cDNA arrays will be used to provide an inexpensive alternative to corroborate the oligonucleotide array approach. In Aim 4, the focus is turned from the analysis of mRNA to DNA sequence variations that may influence transcriptional regulation. Transcriptional promoter sequences in 1,000 candidate genes for mental disorders will be identified and screened for functional variants in a reporter system in cultured cells. The functional variants that show association with expression levels in brain samples will be genotyped in a larger collection of samples containing both cases and controls. Taken together, these alternative approaches complement the other Conte projects, and enhance each other's ability to achieve the overall goal of finding the dysregulated genes and inferring the altered cellular phenotypes in mental disorders.

该项目的目的是确定特定大脑区域的基因表达异常，这些基因表达异常可能是情绪障碍的维持和表现的基础。基本假设是，伴随行为或情绪表型的生物学状态改变可能是由特定脑区基因表达模式的可识别变化引发、维持或修改的，因此，同时监测多个相互连接的脑区中的数万个基因提供了一个很好的机会来定义精神障碍的神经学基础。本申请的重点是开发和应用替代的高通量方法来测量基因表达， 从而克服寡核苷酸微阵列的局限性。在具体目标1中，定量RT-PCR将用于实现更大的灵活性和更少的依赖于基因功能的现有知识，以提供大大提高的特异性，灵敏度和准确性，从而允许常规，深入验证来自其他技术平台的初步发现或来自基因特异性机制假设的候选基因。在目标2中，基因表达的系列分析将用于确保全面性，并提供发现迄今为止在精神障碍中具有未知作用的基因的机会。在目标3中，斑点cDNA阵列将用于提供一种廉价的替代方法来证实寡核苷酸 数组方法在目标4中，重点从mRNA的分析转向可能影响转录调控的DNA序列变异。将在培养细胞的报告系统中鉴定和筛选1,000个精神障碍候选基因中的转录启动子序列的功能变体。显示与脑样品中的表达水平相关的功能变体将在包含病例和对照的较大样品集合中进行基因分型。总而言之，这些替代方法补充了Conte的其他项目，并增强了彼此实现寻找失调基因并推断精神障碍中改变的细胞表型的总体目标的能力。