Design of Genetically Encoded Ca2+ Indicators for in Vivo Application

用于体内应用的基因编码 Ca2 指示剂的设计

• 批准号: 7933652
• 负责人: Michael I. Kotlikoff
• 金额: $ 10.01万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-16 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933652
• 关键词: Address; Animals; Bacterial Artificial Chromosomes; Binding; Biological Process; Calmodulin; Cell Communication; Cell physiology; Cells; Characteristics; Cherry - dietary; Chimeric Proteins; Collaborations; Complex; Development; Disease; Dissection; Endothelial Cells; Event; Fluorescence; Functional disorder; Genetic; Goals; Heart; Image; In Vitro; Kinetics; Laboratories; Link; Mammals; Measurement; Measures; Methods; Modeling; Modification; Molecular; Mus; Nature; Noise; Organ; Performance; Physiology; Proteins; Resolution; Series; Signal Transduction; Skeletal Muscle; Solvents; Spectrum Analysis; Structure; System; Time; Transgenic Mice; absorption; base; cardiogenesis; cholinergic; design; experience; fluorophore; improved; in vivo; intercellular communication; molecular scale; mutant; neuromuscular transmission; novel; prevent; promoter; protein structure function; public health relevance; ratiometric; repaired; response; sensor; thermostability; tool; two-photon; Address; Animals; Bacterial Artificial Chromosomes; Binding; Biological Process; Calmodulin; Cell Communication; Cell physiology; Cells; Characteristics; Cherry - dietary; Chimeric Proteins; Collaborations; Complex; Development; Disease; Dissection; Endothelial Cells; Event; Fluorescence; Functional disorder; Genetic; Goals; Heart; Image; In Vitro; Kinetics; Laboratories; Link; Mammals; Measurement; Measures; Methods; Modeling; Modification; Molecular; Mus; Nature; Noise; Organ; Performance; Physiology; Proteins; Resolution; Series; Signal Transduction; Skeletal Muscle; Solvents; Spectrum Analysis; Structure; System; Time; Transgenic Mice; absorption; base; cardiogenesis; cholinergic; design; experience; fluorophore; improved; in vivo; intercellular communication; molecular scale; mutant; neuromuscular transmission; novel; prevent; promoter; protein structure function; public health relevance; ratiometric; repaired; response; sensor; thermostability; tool; two-photon

DESCRIPTION (provided by applicant): Genetically encoded Ca2+ sensors hold great promise for the dissection of complex physiology in vivo. The ability to make molecular scale measurements in real time in mammals, and to determine lineage-specific signaling events by genetic specification, provides unprecedented experimental power to determine the complex cell-cell communications that underlie normal organ function, and the dysfunction that attends and is the hallmark of disease. A number of laboratories have developed circularly permutated EGFP-Calmodulin/M13 fusion proteins to understand several complex biological processes in vivo, and these tools have begun to provide a novel window on heart development, heart repair, and endothelial cell signaling. While these studies demonstrate the feasibility of real-time, in vivo imaging at the molecular scale in mammals using genetically encoded Ca2+ indicators, limitations of current molecules prevent their comprehensive exploitation. These limitations include less than optimal signal/noise characteristics, a nonlinear Ca2+ response, the limited spectral range of effective probes, and their non-ratiometric nature. The overall goal is to develop improved genetically encoded Ca2+ sensors (GECIs) through the determination of the structural basis of Ca2+ -dependent fluorescence, the development of multiwavelength indicators that provide the ability to quantify Ca2+ signals in vivo, and the creation of red- shifted GECIs that enable studies of cell-cell signaling in vivo. The effort represents an extension of an ongoing collaboration between the laboratories of Dr. Michael Kotlikoff, who has significant experience with the design and function of GECIs, Dr. Holger Sondermann, who is an expert in protein structure and function, and Dr. Warren Zipfel, a biophysicist with expertise in fluorescence photophysics. These studies will address several significant limitations of current molecules and extend the range of studies for which such molecules will be useful. Emphasis will be placed on the optimization of developed sensors for in vivo performance, as determined by their expression in transgenic mice. PUBLIC HEALTH RELEVANCE: This project will produce novel molecules that can be used in vitro in cell systems and in vivo in animals to determine cellular function in the context of disease or organ repair. The proposal will produce novel proteins that will track the function of cells at the molecular level.

描述（由申请人提供）：遗传编码的CA2+传感器对体内复杂生理学的解剖具有很大的希望。在哺乳动物中实时进行分子尺度测量的能力，并通过遗传规范确定谱系特异性的信号传导事件，提供了前所未有的实验能力，以确定基于正常器官功能的复杂细胞 - 细胞通信，以及参加的功能障碍，而疾病的标志也是疾病的标志。许多实验室已经开发了循环排列的EGFP-钙调蛋白/M13融合蛋白，以了解体内的几种复杂的生物学过程，这些工具已开始为心脏发育，心脏修复和内皮细胞信号传导提供新的窗口。尽管这些研究证明了实时的可行性，但使用遗传编码的CA2+指标，在哺乳动物中以分子量表进行体内成像，但当前分子的局限性阻止了它们的全面利用。这些局限性包括少于最佳信号/噪声特性，非线性CA2+响应，有效探针的频谱范围及其非速率量表。总体目标是通过确定CA2+依赖性荧光的结构基础，开发改进的遗传编码的Ca2+传感器（GECIS），开发多波伦长度指标，这些指标提供了在体内量化Ca2+信号的能力，以及启用细胞电池信号的红色转移GECIS的能力。这项工作代表了迈克尔·科特利科夫（Michael Kotlikoff）博士之间正在进行的合作的延伸，他在蛋白质结构和功能方面的专家霍尔格·桑德曼（Holger Sondermann）博士和沃伦·齐普尔（Warren Zipfel）博士（沃伦·齐普尔（Warren Zipfel）博士）和荧光物理学专业知识的生物物理学家沃伦·Zipfel博士（Warren Zipfel）具有丰富的设计和功能。这些研究将解决当前分子的几个重要局限性，并扩展该分子将是有用的研究范围。重点将放在开发的传感器对体内性能的优化，这取决于它们在转基因小鼠中的表达。公共卫生相关性：该项目将产生新的分子，这些分子可以在细胞系统和体内在动物中使用，以确定在疾病或器官修复背景下细胞功能。该建议将产生新型蛋白质，该蛋白质将在分子水平上跟踪细胞的功能。