Unlocking the Hidden Kinome

解锁隐藏的激酶组

• 批准号: 8898229
• 负责人: MICHAEL T MCMANUS
• 金额: $ 32.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8898229
• 关键词: Antibiotics; Apoptosis; Biological; Biology; Bioreactors; Cell Death; Cell model; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Foundations; Future; Genes; Genetic; Genome; Goals; Gold; Guide RNA; Health; Human; Ion Channel; Jurkat Cells; Knowledge; Lead; Libraries; Mammalian Cell; Maps; Methodology; Mission; Nuclear Receptors; Pathway interactions; Phenotype; Phosphotransferases; Positioning Attribute; Process; Proteins; Protocols documentation; Publishing; RNA Interference; Reagent; Reporter Genes; Research Personnel; Resources; Role; Signal Pathway; Signal Transduction; System; Systems Biology; T-Lymphocyte; Technology; Therapeutic; base; cell type; cost effective; dark matter; deep sequencing; gene function; gene interaction; improved; insight; member; next generation; novel; programs; protein function; protein protein interaction; screening; sound; therapeutic target; tool; vector

DESCRIPTION: The goal of this proposal is to further a unique genetic interaction technology to facilitate the unveiling of the functions of the poorly characterized members of the human kinome. Our end goal is to generate game-changing reagents and protocols, and illuminate the "Dark Matter" of the genome. Our strategy is based on ultra-high throughput gene interaction, a technology that we have developed for mammalian cells. Genetic interaction is a gold standard for shedding functional insight into gene function and is complimentary to protein:protein interaction and other standard protocols. Our proposed technologies represent a novel advance on cutting-edge approaches that use CRISPR and RNA interference. We stand ready to engage the project as we have already established a sound technical foundation having recently published the methodology. We describe pooled approaches as a readily scalable technology. This proposal describes our efforts to screen immense numbers of interactions surrounding the human kinome. This will give us insight into functions and pathways for the poorly characterized genes. A major goal is to create customized 'doubles libraries' where each of the 518 human kinase genes are co-ablated against every gene in the genome. This library constitutes millions of gene interaction pairs and is screened at high coverage in bioreactors. Our initial plan is to probe signaling pathways that lead to fundamental phenotypes that relate to loss of proliferation and cell death. However we reserve the option to perform additional screens for specific signaling pathways, using integrated fluorescent:antibiotic reporter genes. These resources are scalable and applicable to nearly any human gene and nearly every cell type. Future screens may scale to explore higher-order interactions, and extend beyond the kinome (i.e., GPCRs, nuclear receptors, and ion channels).

描述：该提案的目的是进一步采取独特的遗传相互作用技术，以促进人类神奇群体特征不佳成员的功能的揭幕。我们的最终目标是生成改变游戏规则的试剂和协议，并阐明基因组的“暗物质”。我们的策略基于超高吞吐量基因相互作用，这是我们为哺乳动物细胞开发的技术。遗传相互作用是将功能性洞悉基因功能的黄金标准，并且与蛋白质相互作用：蛋白质相互作用和其他标准方案。我们提出的技术代表了使用CRISPR和RNA干扰的尖端方法的新型进步。我们已经准备好参与该项目，因为我们已经建立了最近发布该方法的合理技术基础。我们将汇总方法描述为一种易于扩展的技术。该提案描述了我们为筛选人类动物群的大量相互作用的努力。这将使我们深入了解较差的基因的功能和途径。一个主要目标是创建定制的“加倍图书馆”，其中518个人激酶基因中的每个基因都与基因组中的每个基因共同驱动。该库构成数百万个基因相互作用对，并在生物反应器中的高覆盖范围内进行筛选。我们最初的计划是探测导致与丧失增殖和细胞死亡有关的基本表型的信号通路。但是，我们保留使用集成的荧光：抗生素报告基因进行特定信号通路的其他屏幕的选择。这些资源可扩展，几乎适用于任何人类基因和几乎每种细胞类型。未来的筛选可能会扩展以探索高阶相互作用，并延伸超出Kinome（即GPCR，核受体和离子通道）。