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

基本信息

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
项目类别：
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

项目摘要

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）：基因编码的 Ca2+ 传感器对于解剖体内复杂的生理学具有很大的前景。在哺乳动物中实时进行分子尺度测量，并通过遗传规范确定谱系特异性信号传导事件的能力，提供了前所未有的实验能力，可以确定正常器官功能背后的复杂细胞间通讯，以及参与和发生的功能障碍。疾病的标志。许多实验室已开发出循环排列的 EGFP-钙调蛋白/M13 融合蛋白，以了解体内多种复杂的生物过程，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。虽然这些研究证明了使用基因编码的 Ca2+ 指示剂在哺乳动物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碍了其综合利用。这些限制包括低于最佳的信号/噪声特性、非线性 Ca2+ 响应、有效探头的有限光谱范围及其非比例性质。总体目标是通过确定 Ca2+ 依赖性荧光的结构基础、开发能够定量体内 Ca2+ 信号的多波长指示剂以及创建红移信号，开发改进的基因编码 Ca2+ 传感器 (GECI)。 GECI 能够研究体内细胞间信号传导。这项工作是 Michael Kotlikoff 博士（在 GECI 的设计和功能方面拥有丰富经验）、Holger Sondermann 博士（蛋白质结构和功能专家）和 Warren Zipfel 博士实验室之间持续合作的延伸。，一位拥有荧光光物理学专业知识的生物物理学家。这些研究将解决当前分子的几个重大局限性，并扩大此类分子有用的研究范围。重点将放在优化开发的传感器的体内性能，这取决于它们在转基因小鼠中的表达。公共健康相关性：该项目将产生可在细胞系统体外和动物体内使用的新型分子，以确定疾病或器官修复情况下的细胞功能。该提案将产生新的蛋白质，在分子水平上追踪细胞的功能。