Project 5: High-Throughput Analysis of Gene Regulation

这个项目的目的是确定特定大脑区域的基因表达异常，这些异常可能是情绪障碍维持和表现的基础。潜在的假设是，伴随着行为或情绪表型的生物状态的改变很可能是由特定大脑区域中可识别的基因表达模式的变化启动、维持或改变的，因此，同时监测多个相互关联的大脑区域中的数万个基因为确定精神障碍的神经学基础提供了一个极好的机会。这项应用的重点是开发和应用替代的高通量方法来测量基因表达， 从而克服了寡核苷酸微阵列的局限性。在具体目标1中，定量RT-PCR将被用来实现更大的灵活性和对当前基因功能知识的较少依赖，以提供极大的特异性、敏感性和准确性，从而允许常规地、深入地验证来自其他技术平台或来自特定于基因的机械假说的候选基因的初步结果。在目标2中，基因表达的系列分析将被用来确保全面性，并提供机会发现在精神障碍中具有迄今未知作用的基因。在目标3中，斑点cdna阵列将用于提供一种廉价的替代方案来确证寡核苷酸。 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。1,000个精神障碍候选基因的转录启动子序列将被识别，并在培养细胞的报告系统中筛选功能变异。显示与大脑样本中表达水平相关的功能变异将在包含病例和对照的更大样本集合中进行基因分型。综上所述，这些替代方法是对其他CONTE项目的补充，并增强了彼此实现总体目标的能力，即找到失调基因并推断精神障碍中改变的细胞表型。

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.