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

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Ca 2+传感器对于体内复杂生理学的解剖具有很大的希望。在哺乳动物中进行真实的分子尺度测量的能力，以及通过遗传特化确定谱系特异性信号事件的能力，提供了前所未有的实验能力来确定作为正常器官功能基础的复杂细胞-细胞通信，以及伴随疾病的功能障碍。许多实验室已经开发了循环排列的EGFP-Calmodulin/M13融合蛋白，以了解体内几个复杂的生物学过程，这些工具已经开始为心脏发育，心脏修复和内皮细胞信号转导提供新的窗口。虽然这些研究证明了在哺乳动物中使用遗传编码的Ca 2+指示剂在分子尺度上进行实时体内成像的可行性，但当前分子的局限性阻止了它们的全面开发。这些限制包括低于最佳的信号/噪声特性，非线性Ca 2+响应，有效探针的有限光谱范围，以及它们的非比率性质。总体目标是通过确定Ca 2+依赖性荧光的结构基础、开发提供体内定量Ca 2+信号的能力的多波长指示剂以及创建能够在体内研究细胞-细胞信号的红移GECIs来开发改进的遗传编码的Ca 2+传感器（GECIs）。这项工作代表了Michael Kotlikoff博士实验室之间正在进行的合作的延伸，Michael Kotlikoff博士在GECIs的设计和功能方面具有丰富的经验，Holger Sondermann博士是蛋白质结构和功能方面的专家，Warren Zipfel博士是具有荧光物理学专业知识的生物制药学家。这些研究将解决当前分子的几个重要局限性，并扩展这些分子有用的研究范围。重点将放在优化开发的传感器在体内的性能，确定其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，可用于体外细胞系统和动物体内，以确定疾病或器官修复背景下的细胞功能。该提案将产生新的蛋白质，在分子水平上跟踪细胞的功能。