A New Modular Tool Set for Live Imaging and Manipulating the Nervous System

描述（由申请人提供）：用于实时成像和操纵神经系统的新模块化工具集项目总结。神经系统包含了人体中最复杂、最多样化的细胞类型。现代成像方法和基于绿色荧光蛋白（GFP）的基因编码报告，使得探索神经系统发育、功能和发病机制中的一些动态过程成为可能。然而，神经系统的复杂性使得精确地靶向特定神经元的表达变得困难。这项提议的目的是创造和测试全新一代的基因编码、模块化荧光分子的原型，用于标记和成像特定的细胞类型，并用于操纵它们表达的基因。许多神经系统细胞类型的特异性启动子是不可用的，这阻碍了基因进入这些细胞。使用创新的组合策略，该项目将提供对这些细胞的访问，使研究人员能够特异性靶向基因表达，并获得对信号转导，蛋白质-蛋白质相互作用以及神经元功能和发病机制的动态过程的新见解。目标之一是创造一对模块化的标记蛋白，Fly和Hook，它们被亮氨酸拉链绑在一起。这种模块化系统将提供对迄今为止无法进行标记和纯化的神经元的访问。目标二是创建一个双功能模块化系统，用于标记神经元和通过产生荧光和Cre重组酶活性的互补蛋白质操纵其基因组。神经系统的发育和功能，以及神经元在疾病和损伤中的死亡，是一个动态的过程，人们对其了解甚少。该项目的目标是开发新的荧光分子，用于基因标记和操纵活脑中特定神经元的基因表达。如果这个项目是可行的，这些分子将导致对癫痫、中风和阿尔茨海默氏症等神经系统疾病发病机制的动态过程有新的认识。

DESCRIPTION (provided by applicant): A New Modular Tool Set for Live Imaging and Manipulating the Nervous System Project summary. The nervous system contains the most complex and heterogeneous set of cell types in the body. Modern approaches in imaging, and genetically encoded reporters based on the Green Fluorescent Protein (GFP), have made it possible to explore some of the dynamic processes involved in the development, function, and pathogenesis of the nervous system. However, the complexity of the nervous system has made it difficult to precisely target expression to particular sets of neurons. The objective of this proposal is to create and test prototypes of an entirely new generation of genetically encoded, modular fluorescent molecules for labeling and imaging specific cell types and for manipulating the genes that they express. Specific promoters for many cell types of the nervous system are not available, barring genetic access to these cells. Using an innovative combinatorial strategy, this project will provide access to these cells and enable researchers to specifically target gene expression and gain new insight into signal transduction, protein-protein interaction, and the dynamic processes of neuronal function and pathogenesis. Aim one is to create a modular pair of labeling proteins, the Fly and the Hook, that are tied together by leucine zippers. This modular system will provide access to neurons that have eluded labeling and purification to date. Aim two is to create a bi-functional, modular system for both labeling neurons and manipulating their genomes with complementing proteins that produce both fluorescence and Cre recombinase activity. A New Modular Tool Set for Live Imaging and Manipulating the Nervous System Project Narrative The development and function of the nervous system, as well as the death of neurons in disease and injury, are dynamic processes that are poorly understood. The goal of this project is to develop new fluorescent molecules for genetically labeling and manipulating gene expression in specific neurons in the living brain. If this project is feasible, these molecules would lead to new understanding of the dynamic processes that underlie the pathogenesis of neurological diseases such as epilepsy, stroke and Alzheimer's.