GENOMIC RESPONSES TO HORMONE SIGNALING

只有所有编码基因的一个子集在任何给定的细胞中表达，并且

Only a subset of all encoded genes is expressed in any given cell, and the levels and timing of expression govern cellular development, differentiation, function and physiology. Here we describe a method for the simultaneous monitoring of the expression levels of many genes in parallel. The method involves the determination of the relative concentrations of mRNAs based on hybridization of entire mRNA populations to high-density arrays of synthetic oligonucleotides (GeneChips TM). The arrays used for the experiments proposed here contain more than 65,000 different 25-mer oligonucleotides of defined sequence in an area of 1.6 cm 2. In Phase I, we will: 1) Design a set of arrays (GeneChip TM) for the simultaneous quantitation of over 6500 human genes (Hum6000) from GenBank and the public EST database (dbEST). 2) Demonstrate that the method is sensitive, specific and quantitative. 3) Use the Hum6000 chip to determine the mRNAs dynamically regulated by three hormone receptors (beta-2 Adrenergic, CCR5, Oxytocin) representing three different signaling pathways. In Phase II we will use the Hum6000 chip to analyze the mRNAs that are dynamically regulated by a large panel of receptors, and pharmacologic probes. We will also confirm a subset of the findings from the Hum6000 chip by using Northern analysis, Western blots, and gene expression markers. PROPOSED COMMERCIAL APPLICATION: The quantitative monitoring of expression levels for large numbers of genes should prove valuable in elucidating gene function, exploring the causes and mechanisms of disease, and for the discovery of potential therapeutic and diagnostic targets. As the body of genomic information grows, highly parallel methods of the type described here provide an efficient and direct way to use sequence information to elucidate important hormone, drug and metabolic pathways. Development of the GeneChip technology as a tool to monitor genome-wide expression profiles has multifold biomedical, pharmaceutical and agricultural applications. Revenue will be derived from the sale of the GeneChip system (fluidics station and scanner) and custom GeneChips Studies with hormone receptors will identify genes that will be potential new targets for drug development. The regulatory regions of hormonally activated genes could also be used to develop rapid, non-radioactive reporter assays for high- throughput screening of drugs that activate hormone receptors.