Novel Approaches to Functional Genomics

描述(由申请人提供)：数量惊人的新技术 目前正在开发，将塑造生物医学研究在 后基因组时代。其中许多方法属于基因组学领域， 将允许使用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。其他内容 技术将强调功能基因组学，并将利用DNA 用微阵列探测大约100,000个哺乳动物基因在 来自生理和疾病状态的各种细胞和组织。最后， 仪器仪表领域的巨大技术进步催生了 蛋白质组学，对蛋白质和蛋白质修饰进行大量研究 在基因组信息的背景下进行扩展。 这项提议描述了一种新的界面技术的发展 在功能基因组学和蛋白质组学之间。这项技术涉及 表达载体DNA在细胞表面的物理附着 在文化中成长。然后将培养中的细胞施加到处理过的表面上， 那些应用于含有表达载体的“斑点”的细胞是 转基因，表达编码蛋白。STEP(表面转染法和 Expression协议)有可能研究数十个 数以千计的蛋白质来自一个单一的cdna表达载体 显微镜载玻片。分步转染法可方便地应用于DNA微阵列 打印幻灯片的生成和蛋白质定量的格式 功能。 提出的研究是在三个具体目标下进行的。所有这些都是 具体目标代表可以开发STEP有效性的实例 在一个具有科学意义的背景下。第一个具体目标是发展 分步技术到可以用来筛查突变的 蛋白激酶文库，以定义蛋白的结构域 激活剂。第二个特定目标将使用步骤来优化反义 降低蛋白激酶活性的寡核苷酸策略 被认为在细胞增殖中起着核心作用。第三个具体目标 将定义一组小的、已定义的蛋白激酶的相互作用， 功能步进分析中的转录因子和基因调控元件。 在完成这三个具体目标后，步法技术将是 充分发展到科学界可以将其应用于相关的 研究问题。

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.