Novel Approaches to Functional Genomics

功能基因组学的新方法

基本信息

批准号：
6616714
负责人：
MICHAEL D UHLER
金额：
$ 31.38万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2005-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): An astounding number of novel techniques are currently being developed that will shape biomedical research in the post-genomic era. Many of these approaches fall into the area of genomics and will allow the prediction of function of genes using sophisticated comparisons of protein sequence and structure between various organisms. Additional techniques will emphasize functional genomics and will take advantage of DNA microarrays to probe the expression of the estimated 100,000 mammalian genes in various cells and tissues from both physiological and diseased states. Finally, great technological advances in instrumentation have given rise to the field of proteomics, where proteins and protein modifications are studied on a large scale in the context of genomic information. This proposal describes the development of a novel technique at the interface between functional genomics and proteomics. This technique involves the physical attachment of expression vector DNA to surfaces on which cells can be grown in culture. Cells in culture are then applied to the treated surfaces and those cells applied to the "spots" containing expression vector are transfected, expressing the encoded protein. The STEP (Surface Transfection and Expression Protocol) has the potential to study the function of tens of thousands of proteins derived from cDNA expression vectors on a single microscope slide. STEP transfection can be readily adapted to DNA microarray formats for generation of printed slides and for quantitation of protein function. The research proposed is organized under three specific aims. All of these Specific Aims represent instances where the efficacy of STEP can be developed in a scientifically meaningful context. The first Specific Aim is to develop the STEP technique to the point where it can be used to screen a mutational library of a protein kinase in order to define the structural domains of the kinase. The second Specific Aim will use STEP to optimize an antisense oligonucleotide strategy to reduce the enzyme activity of a protein kinase thought to play a central role in cell proliferation. The third Specific Aim will define the interactions of a small, defined set of protein kinases, transcription factors and gene regulatory elements in functional STEP assays. Upon completion of these three Specific Aims, the STEP technique will be sufficiently developed for the scientific community to apply it to related research problems.

描述（由申请人提供）：数量惊人的新技术目前正在开发中，这将影响生物医学研究后基因组时代。其中许多方法属于基因组学领域将允许通过复杂的比较来预测基因的功能不同生物体之间的蛋白质序列和结构。额外的技术将强调功能基因组学并利用 DNA 微阵列来探测大约 100,000 个哺乳动物基因的表达生理和疾病状态下的各种细胞和组织。最后，仪器仪表领域的巨大技术进步催生了蛋白质组学，大规模研究蛋白质和蛋白质修饰基因组信息范围内的规模。该提案描述了接口新技术的开发介于功能基因组学和蛋白质组学之间。该技术涉及将表达载体 DNA 物理附着到细胞可以在其上的表面在文化中成长。然后将培养的细胞施加到经过处理的表面上那些应用于含有表达载体的“点”的细胞是转染，表达编码的蛋白质。 STEP（表面转染和表达协议）具有研究数十种功能的潜力来自 cDNA 表达载体的数千种蛋白质显微镜载玻片。 STEP 转染可轻松适应 DNA 微阵列用于生成打印幻灯片和用于蛋白质定量的格式功能。拟议的研究根据三个具体目标进行组织。所有这些具体目标代表了可以开发 STEP 功效的实例在具有科学意义的背景下。第一个具体目标是发展 STEP 技术可用于筛选突变蛋白激酶文库，以确定其结构域激酶。第二个特定目标将使用 STEP 来优化反义降低蛋白激酶酶活性的寡核苷酸策略被认为在细胞增殖中发挥核心作用。第三个具体目标将定义一小部分明确的蛋白激酶的相互作用，功能性 STEP 测定中的转录因子和基因调控元件。完成这三个具体目标后，STEP 技术将被足以让科学界将其应用于相关领域研究问题。