Creation of a Versatile Proteomic Resource in Murine Embryonic Stem Cells

在小鼠胚胎干细胞中创建多功能蛋白质组资源

• 批准号: 8832876
• 负责人: Vaidyanathan Subramaniam
• 金额: $ 30.05万
• 依托单位: POWERHOUSE PROTEOMIC SYSTEMS, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8832876
• 关键词: Am 80; Applied Research; Basic Science; Biological Assay; Cell Line; Cells; DNA; Detection; Development; Disadvantaged; ES Cell Line; Effectiveness; Embryo; Event; Excision; Exons; Fluorescence; Fluorescence Microscopy; Future; Gene Proteins; Genes; Genomics; Goals; Green Fluorescent Proteins; In Situ; Libraries; Life; Methods; Molecular; Molecular Profiling; Mus; Phase; Preclinical Drug Evaluation; Proteins; Proteome; Proteomics; Reporter; Reporting; Research; Research Personnel; Resources; Series; Signal Transduction; System; Techniques; Technology; Testing; Therapeutic Agents; Time; TimeLine; Toxic effect; Variant; Vertebral column; Viral; Work; base; design; embryonic stem cell; human disease; improved; in vivo; next generation sequencing; novel; novel strategies; protein expression; public health relevance; screening; stem; tool; vector

DESCRIPTION (provided by applicant): The ultimate goal of this project is to create a novel proteomic resource that will enable the real-time expression profiling of proteins in live murine embryonic stem (mES) cells and in differentiated cells derived therefrom. The resource will be in the form of an annotated bank of protein-trap mES cell lines, each of which will carry a single tagged gene. Fluorescent signal in these lines will report the expression and subcellular localization of the protein product(s) of the tagged gene in each line. Such a library of clonal fluorescently- tagged lines can be used, in conjunction with quantitative fluorescence microscopy in multiwell live-cell arrays, to study thousands of proteins in live cells, in real tim, under various experimental conditions. In a separate effort, we have initiated the development of such a bank of fluorescent protein-trap mES cell lines. However, the technology we have used so far will only allow us to detect and isolate protein-traps in genes that are actively expressed in mES cells; this limits the breadth and versatility of the library because the bulk of the proteome (>80%) is estimated to be effectively silent in mES cells. In order to create a truly broad proteomic library, we have devised a method that will allow us to identify and isolate protein-traps in genes that are silent in mES cells. Further, in this new approach, we will also add a more versatile protein tag (FAP-tag) so as to greatly increase the sensitivity of fluorescence detection in (and therefore the overall functionality of) the protein-trap mES library This Phase I project is designed to construct the requisite DNA vectors, to test their efficacy at identifying protein-traps in silent genes, and to assess their scope and suitability for use in a future large-scale protein-trapping effort.

描述（由申请人提供）：该项目的最终目标是创建一种新型的蛋白质组学资源，该资源将使蛋白质在活鼠胚胎词干（MES）细胞（MES）细胞中以及从中得出的分化细胞中进行实时表达分析。该资源将以带注释的蛋白质陷阱细胞系的注释库的形式，每个蛋白质陷阱细胞系将携带一个标记的基因。这些线中的荧光信号将报告每条线中标记基因的蛋白质产物的表达和亚细胞定位。这种克隆荧光标记线的库可以与多井活细胞阵列中的定量荧光显微镜一起使用，以在各种实验条件下在实际TIM中研究活细胞中的数千种蛋白质。 在另一项努力中，我们启动了这种荧光蛋白陷阱MES细胞系的发展。但是，到目前为止我们使用的技术只能使我们能够在MES细胞中积极表达的基因中检测和分离蛋白质陷阱。这限制了库的广度和多功能性，因为估计在MES细胞中估计蛋白质组的大部分（> 80％）有效地保持沉默。为了创建一个真正广泛的蛋白质组学库，我们设计了一种方法，它将允许我们识别和分离MES细胞中沉默的基因中的蛋白质陷阱。此外，在这种新方法中，我们还将添加一个更通用的蛋白质标签（FAP-TAG），以极大地提高荧光检测的敏感性（以及因此）蛋白质捕集MES库中该阶段I项目旨在构建必要的DNA载体，以测试其在众多蛋白质中的效率，并评估其在稳固的基因中的效率，并在稳定的蛋白质中进行效率，并构建其效率。蛋白质捕获的工作。