A Novel Approach for Real-Time Proteomics in Live Cells

活细胞实时蛋白质组学的新方法

• 批准号: 7477488
• 负责人: Vaidyanathan Subramaniam
• 金额: $ 20万
• 依托单位: POWERHOUSE PROTEOMIC SYSTEMS, LLC
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7477488
• 关键词: Address; Animals; Biological Assay; Biological Markers; Biological Sciences; Cardiac Myocytes; Cardiovascular Diseases; Cell Line; Cells; Clonal Expansion; Condition; Custom; Detection; Development; Diabetes Mellitus; Diagnostic tests; Disease; ES Cell Line; Eligibility Determination; Embryo; Event; Exons; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Gene Proteins; Generations; Genes; Goals; Histocompatibility Testing; Libraries; Life; Location; Methods; Mitochondrial Proteins; Molecular; Molecular Profiling; Monitor; Mus; Neurodegenerative Disorders; Numbers; Phase; Phase I Clinical Trials; Preclinical Drug Evaluation; Proteins; Proteome; Proteomics; Protocols documentation; Reporter; Research Personnel; System; Techniques; Technology; Testing; Therapeutic Agents; Time; Transgenic Mice; base; desire; embryonic stem cell; enhanced green fluorescent protein; human disease; interest; novel; novel diagnostics; novel strategies; promoter; protein expression; stem; therapeutic target; vector

DESCRIPTION (provided by applicant): The ultimate aim of this project is to develop a novel proteomic system that will enable the real-time expression profiling of proteins in live murine embryonic stem (mES) cells and, ultimately, in differentiated cells derived therefrom. In the first step, a library of clonal mES cell lines will be established, each of which will harbor a single fluorescently-tagged protein. The fluorescence in each cell of a particular clonal line will serve as a reporter for the expression level and subcellular localization of the tagged protein in that cell line. This library of clonal fluorescently-tagged lines can be subjected to quantitative fluorescence microscopy in multi- well live-cell arrays, to enable the simultaneous real-time expression profiling of multiple proteins in live cells under various culture conditions. This Phase I study will be focused primarily on developing as extensive a library in mES cells as is practically possible. Such a 'master' library of fluorescently-tagged mES cell lines can also be manipulated in culture into specific tissue-types such as cardiomyocytes, to create a powerful new system for biomarker discovery and drug-screening relevant to economically important diseases such as cardiovascular disease. Further, these mES cells can also be used for the generation of transgenic mice as and when desired, to extend such studies to live animals. The ultimate aim of this project is to develop a system that will enable researchers to monitor the location and levels of hundreds of proteins simultaneously in living cells. Such a system will be useful in understanding the molecular basis of a number of human diseases, such as cardiovascular disease, neurodegenerative disease and diabetes. Further, such a system can be applied to the development of novel diagnostic tests, identification therapeutic targets and the creation of powerful drug-screening platforms to identify potential therapeutic agents that may cure these diseases.

描述（由申请人提供）：该项目的最终目的是开发一种新型的蛋白质组学系统，该系统将使蛋白质在活鼠胚胎（MES）细胞（MES）细胞中的实时表达分析，并最终在其衍生的区分细胞中。在第一步中，将建立一个克隆MES细胞系的库，每个库将带有单个荧光标记的蛋白质。特定克隆线的每个细胞中的荧光将作为该细胞系中标记蛋白的表达水平和亚细胞定位的报告。在多井活细胞阵列中，该克隆荧光线的库可以进行定量荧光显微镜，以使在各种培养物条件下活细胞中多个蛋白质的同时实时表达分析。这一I阶段研究将主要集中于在MES细胞中以广泛的形式开发作为广泛的图书馆。这种荧光标记的MES细胞系的“主”库也可以在培养中被操纵到特定的组织类型（例如心肌细胞）中，以创建一个强大的新系统，以实现生物标志物发现和与经济上重要疾病（如心血管疾病）相关的药物筛查。此外，这些MES细胞也可以根据需要的方式用于生成转基因小鼠，以将这些研究扩展到活动物。该项目的最终目的是开发一个系统，该系统将使研究人员能够同时监测活细胞中数百种蛋白质的位置和水平。这样的系统将有助于理解许多人类疾病的分子基础，例如心血管疾病，神经退行性疾病和糖尿病。此外，这种系统可以应用于新的诊断测试，识别治疗靶标和创建强大的药物筛查平台的开发中，以鉴定可能治愈这些疾病的潜在治疗剂。