Biological Oscilloscopes Spatio-Temporal Metabolomics

生物示波器时空代谢组学

• 批准号: 7107128
• 负责人: John D. York
• 金额: $ 176.7万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7107128
• 关键词: NIH Roadmap Initiative tag; bioimaging /biomedical imaging; biological signal transduction; bioperiodicity; biosensor device; biotechnology; cell biology; cell line; cooperative study; embryo /fetus cell /tissue; fluorescence microscopy; fluorescent dye /probe; genetic manipulation; hippocampus; image processing; laboratory rat; metabolomics; nanotechnology; protein engineering; technology /technique development; time resolved data

DESCRIPTION (provided by applicant): The goal of this project is develop a system for real-time in vivo spatiotemporal quantification of metabolic fluctuations at the cellular level. To achieve this, we combines four key technologies: i) genetically encoded fluorescent nanosensor proteins (GENAs) that report on specific metabolites; ii) protein engineering techniques that permit the systematic construction of nanosensors for a large number of metabolites of interest; iii) quantitative real-time fluorescent microscopy visualization techniques that permit these nanosensors to be used in living cells; iv) genetically engineered cell lines that perturb signal transduction pathways by gain-of-function (protein over-expression) and loss-of-function (deletion libraries, RNAi). Our approach is generalizable, scalable, sensitive and dynamic enabling time-resolved measurements in the millisecond range. Using these reagents, it is anticipated that over 10,000 signaling state perturbations will be analyzed for metabolic changes. It is anticipated that many of the signal pathway perturbations and alterations in metabolic profiles will provide a large database of correlative changes, recapitulation and diagnostic potential of disease states and novel biological discovery of signaling networks. We have assembled a research team consisting of four groups that each bring part of the required skills to the project. The Meyer group (Stanford University) has a long-standing involvement in the development of GENAs, and has also been heavily involved in the development of the fluorescence microscopy instrumentation for metabolic visualization. Furthermore, the Meyer group is also involved in development of reagent sets for metabolic perturbations. The Frommer group (Carnegie Institute of Washington) has developed GENAs for sensing of sugars, and has demonstrated that these can be used for in vivo metabolic imaging. The York group (Duke University) has been deeply involved in the genetic manipulation, metabolism and discovery of intracellular signaling pathways. The Hellinga group (Duke University) has been instrumental in the development and experimental validation of computational protein design tools for the radical manipulation of ligand-binding specificities. At the end of the project period, we aim to deliver i) nanosensor toolkits; ii) engineered cell line toolkits; iii) spatiotemporal imaging datasets of metabolic fluctuations as a function of genetic perturbations and exogenous changes in cellular environment.

描述（由申请人提供）：该项目的目的是开发一种用于实时体内时空定量代谢波动在细胞水平上的系统。为了实现这一目标，我们结合了四个关键技术：i）报告特定代谢物的遗传编码荧光纳米传感器蛋白（GenAS）； ii）蛋白质工程技术允许对大量感兴趣的代谢产物进行系统的纳米传感器； iii）定量的实时荧光显微镜可视化技术，允许这些纳米传感器用于活细胞； iv）基因工程的细胞系，通过功能获得（蛋白质过表达）和功能丧失（删除文库，RNAi）扰动信号转导途径。我们的方法是可推广，可扩展，敏感和动态的，启用了毫秒范围内的时间分辨测量。使用这些试剂，预计将分析10,000多个信号状态扰动以进行代谢变化。可以预料，许多信号途径扰动和代谢概况的改变将为疾病状态的相关性变化，概括和诊断潜力以及信号网络的新生物学发现提供大量的相关性变化数据库。我们组建了一个由四个小组组成的研究团队，每个小组都将部分所需技能带入了该项目。迈耶集团（斯坦福大学）长期参与了基因的发展，并且也很大程度上参与了荧光显微镜仪器的发展以进行代谢可视化。此外，迈耶组还参与了代谢扰动的试剂集的开发。 Frommer Group（华盛顿州卡内基研究所）开发了用于感知糖的基因，并证明这些基因可用于体内代谢成像。约克集团（杜克大学）一直参与遗传操作，代谢和细胞内信号通路的发现。 Hellinga Group（杜克大学）在开发和实验验证的计算蛋白设计工具方面发挥了作用，用于对配体结合特异性进行根本性操纵。在项目期结束时，我们旨在提供i）纳米传感器工具包； ii）工程的单元线工具包； iii）代谢波动的时空成像数据集是遗传扰动和细胞环境中外源变化的函数。