Project 5: High-Throughput Analysis of Gene Regulation

点击翻译按钮获取中文摘要

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.