Development of Protein-based Voltage Probes

基于蛋白质的电压探针的开发

• 批准号: 8827112
• 负责人: Vincent A Pieribone
• 金额: $ 52.5万
• 依托单位: JOHN B. PIERCE LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827112
• 关键词: Action Potentials; Behavior; Brain; Calcium; Cell membrane; Cells; Characteristics; Circadian Rhythms; Communities; Complementary DNA; DNA; Data; Detection; Development; Devices; Drosophila genus; Electrodes; Electrophysiology (science); Evolution; Exhibits; Experimental Models; Fluorescence; Fluorescent Probes; Future; Genetic; Goals; Image; In Vitro; Kinetics; Length; Libraries; Light; Measures; Membrane Potentials; Methods; Microelectrodes; Molecular; Monitor; Mus; Mutation; Neurons; Noise; Optical Methods; Optics; Performance; Positioning Attribute; Preparation; Process; Production; Property; Proteins; Publications; Publishing; Reagent; Research; Research Personnel; Sampling; Scientist; Screening Result; Series; Signal Transduction; Site; Speed; Testing; Transfection; Transgenic Mice; Transgenic Organisms; Validation; Visual; adeno-associated viral vector; base; cDNA Library; calcium indicator; cell type; design; dissemination research; fluorescence microscope; fly; improved; in vivo; novel; novel strategies; olfactory bulb; particle; patch clamp; plasmid DNA; public health relevance; research study; response; scaffold; screening; sensor; tau Proteins; two-photon; voltage; voltage/patch clamp

 DESCRIPTION (provided by applicant): The use of genetically encoded fluorescent activity probes represent the most advanced method to monitor the electrical activity of networks of neurons without using electrodes. While genetically encoded calcium indicators have been evolved to produce robust signals in a variety of different neuronal preparations, fluorescent probes of membrane potential have not been well evolved. Current voltage probes, while finally in expanded use, will need considerable improvement if the goal of recording the activity of a large number of neurons simultaneously in vivo is to be achieved. The goal of this project is to discover protein-based fluorescent voltage probes with signal to noise characteristics that allow routine optical recording of action potentials from single cortical neurons in vivo. We are seeking probes with significantly improved signal to noise characteristics, red-shifted fluorescence spectra, faster on and off rates and better plasma membrane expression. This project brings together leading genetic probe scientists to: design new probe scaffolds, robotically screen large incrementally modified libraries of probes, identify probes with improved response properties, then validate these new probes under standardized experimental conditions in a range of 'real world' neuronal preparations with increasing levels of complexity. Finally, we will make all probe reagents (i.e. plasmid DNA, AAV particles, transgenic flies, etc.) as well as supporting validation data readily available to the research community.

 描述（由申请人提供）： 使用基因编码的荧光活性探针代表了在不使用电极的情况下监测神经元网络的电活动的最先进的方法。虽然遗传编码的钙指示剂已经发展到在各种不同的神经元制剂中产生强大的信号，但膜电位的荧光探针还没有得到很好的发展。电流电压探针，而最终在扩大使用，将需要相当大的改进，如果要实现的目标，同时记录大量的神经元在体内的活动。本项目的目标是发现基于蛋白质的荧光电压探针，其具有信噪比特性，允许常规光学记录体内单个皮层神经元的动作电位。我们正在寻求 具有显著改善的信噪比特征、红移的荧光光谱、更快的开和关速率和更好的质膜表达的探针。该项目汇集了领先的遗传探针科学家：设计新的探针支架，机器人筛选大型增量修饰的探针库，识别具有改进响应特性的探针，然后在标准化实验条件下在一系列复杂程度不断增加的“真实的世界”神经元制剂中验证这些新探针。最后，我们将使所有探针试剂（即质粒DNA、AAV颗粒、转基因果蝇等）以及支持验证 研究界可随时获得的数据。