High Throughput PCR-Based Gene Expression Screening

描述(由申请人提供)： 我们癌症遗传学知识库的指数增长导致了大量基因、蛋白质和途径的确定，这些基因、蛋白质和途径可能在癌症发生中发挥核心作用，并可能成为治疗干预的潜在靶点。现在的挑战是深入研究不同化合物和化合物类别如何影响这些基因。可以在基因表达水平上跟踪大量不同的细胞活动，包括详细跟踪细胞和组织对引入药理学试剂的反应的能力。因此，基因表达分析可以用来提供对现有和潜在疗法的靶上和靶外机制活动的详细和广泛的洞察。这里提出了一种新的面向生产的、高通量的、基于PCR的基因表达分析方法的演示和开发。该方法建立在聚合酶链式反应的基本优势的基础上，并增加了高水平的多重，每个聚合酶链式反应增加了15-35个基因。 这项建议的主要目的是开发和证明多重聚合酶链式反应方法作为复合库基因表达筛选的价值。为了实现这一目标，我们建议(A)开发和验证3种多重分析(遗传毒性、肾毒性和HDAC靶向抗肿瘤活性)，(B)开发高通量筛选方案，以及(C)通过筛选Gen-Plus和NCI化学多样性集并对HDAC靶向候选进行先导优化研究来展示其实用性。制定的方案和分析方法以及这些示范筛查研究的数据将公之于众。

DESCRIPTION (provided by applicant): The exponential growth in our knowledgebase of cancer genetics has led to the identification of a large array of genes, proteins and pathways that potentially play a central role in the carcinogenesis and may be potential targets for therapeutic intervention. The challenge now is to delve into how different compounds and compound classes' influence these genes. An enormous number of different cellular activities can be tracked at the gene expression level, including the ability to track in detail the response of cells and tissues to the introduction of a pharmacological agent. For this reason, gene expression analysis can be used to provide detailed and broad insight into the on and off-target mechanistic activities of existing and potential therapeutics. Proposed here is the demonstration and development of a new production-oriented, high throughput, PCR-based approach to gene expression analysis. The approach builds off of the fundamental strengths of PCR and adds a high level of multiplexing, 15-35 genes per PCR reaction. The principal goal of this proposal is to develop and demonstrate the value of a multiplexed PCR approach for use as a compound library gene expression screen. In order to achieve this goal we propose to (a) develop and validate 3 multiplex assays (genotoxicity, nephrotoxicity and HDAC-targeting antitumor activity), (b) develop protocols for high throughput screening, and (c) demonstrate the utility by screening the Gen- Plus and NCI chemical diversity set and performing a lead optimization study on HDAC-targeting candidates. The protocols and assays developed and the data from these demonstration screening studies will be made publicly available.