Unlocking the Hidden Kinome

解锁隐藏的激酶组

• 批准号: 9114166
• 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 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9114166
• 关键词: Antibiotics; Apoptosis; Biological; Biology; Bioreactors; CRISPR interference; CRISPR/Cas technology; Cell Death; Cell model; Cell physiology; Cells; 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; targeted treatment; 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：这项提议的目标是进一步发展一种独特的基因相互作用技术，以促进揭示人类亲属组中特征不佳的成员的功能。我们的最终目标是产生改变游戏规则的试剂和方案，并阐明基因组的“暗物质”。我们的策略是基于超高通量的基因相互作用，这是我们为哺乳动物细胞开发的一项技术。基因相互作用是了解基因功能的黄金标准，是对蛋白质：蛋白质相互作用和其他标准方案的补充。我们提出的技术代表了使用CRISPR和RNA干扰的尖端方法的新进展。我们随时准备参与该项目，因为我们已经建立了良好的技术基础，最近公布了该方法。我们将池化方法描述为一种易于扩展的技术。这项建议描述了我们为筛选围绕人类亲属组的大量相互作用所做的努力。这将使我们深入了解特征不佳的基因的功能和途径。一个主要目标是创建定制的“双重库”，其中518个人类激酶基因中的每一个都与基因组中的每一个基因共同消融。该文库包括数百万个基因相互作用对，并在生物反应器中进行高覆盖率的筛选。我们最初的计划是探索导致与增殖丧失和细胞死亡相关的基本表型的信号通路。然而，我们保留了使用整合的荧光：抗生素报告基因对特定信号通路进行额外筛选的选项。这些资源是可扩展的，适用于几乎任何人类基因和几乎所有细胞类型。未来的屏幕可能会扩展到探索更高级别的相互作用，并延伸到动态组之外(即GPCRs、核受体和离子通道)。