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 蛋白质序列和结构的差异。额外 技术将强调功能基因组学， 微阵列，以探测估计100，000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， 仪器仪表的巨大技术进步已经引起了 蛋白质组学，其中蛋白质和蛋白质修饰研究在一个大的 在基因组信息的背景下。 该提案描述了一种新的技术在接口的发展 功能基因组学和蛋白质组学之间的联系该技术涉及 将表达载体DNA物理附着到细胞可在其上 在文化中成长。然后将培养的细胞施加到处理的表面上， 那些应用于含有表达载体的“斑点”的细胞 转染，表达编码的蛋白质。STEP（Surface Transfection） 表达方案）具有研究数十种基因的功能的潜力。 来自cDNA表达载体的数千种蛋白质在单个 显微镜载玻片。STEP转染可以很容易地适用于DNA微阵列 用于生成打印载玻片和定量蛋白质的格式 功能 拟议的研究是根据三个具体目标组织的。所有这些 具体目标代表STEP的功效可以得到发展的实例 有科学意义的背景下。第一个目标是发展 STEP技术可以用来筛选突变， 蛋白激酶的结构域，以确定蛋白激酶的结构域。 激酶。第二个特定目标将使用STEP优化反义核酸， 降低蛋白激酶的酶活性的寡核苷酸策略 被认为在细胞增殖中起核心作用。第三个具体目标 将定义一小部分蛋白激酶的相互作用， 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 科学界已充分开发，可将其应用于相关领域， 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.