Novel transgenic mice for tracing and electrophysiology of stressed neurons

用于应激神经元追踪和电生理学的新型转基因小鼠

基本信息

批准号：
8361031
负责人：
TSONWIN HAI
金额：
$ 24.06万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8361031
关键词：
Acute Address Affect Afferent Neurons Alleles Basic Science Behavior assessment Biochemical Genetics Brain Concussion Breeding Cell physiology Cells Cellular Stress Cellular Stress Response Characteristics Chimeric Proteins Chronic Core Facility Demyelinations Disease Electrophysiology (science)Event Exhibits Gated Ion Channel Gene Expression Generations Genes Genetic Goals Hand Health Image In Vitro Individual Injection of therapeutic agent Injury Ischemia Knockout Mice Label Lasers Life Light Long-Term Effects Microscopy Modeling Motor Neurons Mus Nerve Crush Neurologic Neurons Nuclear Operative Surgical Procedures Optics Pain Pathology Peripheral Nervous System Physiological Play Population Process Production Promoter Regions Proteins Reporter Research Signal Transduction Stress Synapses System Tamoxifen Time Tissues Transgenes Transgenic Mice Transgenic Organisms Trauma Venus activating transcription factor 3 analog base biological adaptation to stress cell injury chemotherapeutic agent chronic pain in vivo injured innovation light gated mouse model nervous system disorder neurological pathology novel optogenetics recombinase response response to injury stressor tissue fixing tool

项目摘要

DESCRIPTION (provided by applicant): Numerous neurological diseases and pathologies include, or are hypothesized to include, cellular injury and/or stress as part of the mechanism. Experimental approaches to examine these mechanisms, or even determine if they are indeed at play in certain conditions, are generally limited. They include post-mortem analyses of known stress-response genes/molecules, in vivo pharmacological manipulations which are often systemic, or functional assessments/manipulations which often cannot separate stressed from non-stressed neurons. Ideally, it would be possible to determine, a priori on a cell-by-cell basis, which neurons in a mixed population were exhibiting a cell-stress response. Further, since some stress responses, or at least some components of the stress response, are only transiently expressed even though the overall stress responses may have a cumulative effect on the cell, it would be highly useful to have an a priori indication of which neurons had exhibited a stress response at some point in the past. It would also be highly useful to be able to selectively assess the cellular and inter-cellular functionality of the stressed neurons. To these ends we propose to generate new functional-reporter transgenic mouse lines which will incorporate these characteristics. The reporters will be driven by the promoter region of Activating Transcription Factor 3 (ATF3), a "hub" protein involved in a variety of cellular stress responses. Generation of the mice will be via BAC-transgenes in order to preserve function of the native ATF3 alleles, which are necessary for certain cellular processes. The BAC transgene with the ATF3 locus will drive production of the light-gated ion channel channelrhodopsin-2 (ChR2) fused to a fluorescent protein. One model will have the reporters produced in an analogue fashion (i.e., to reflect native ATF3 expression). A second model will use an inducible Cre-recombinase system to permanently "switch-on" reporter production, thus allowing, at any later time, identification of neurons that previously expressed a stress response. These functional-reporter models will allow offline anatomical assessments, FACS or laser-capture separations, fate-tracing of previously-stressed neurons, and in vivo or in vitro functional assessments specifically of stressed neurons (i.e., those expressing ChR2). PUBLIC HEALTH RELEVANCE: This project is intended to generate research tools, specifically 2 new lines of transgenic mice that will produce functional and anatomical reporters in response to cellular stress. These lines would significantly enhance basic science approaches to understand neurological processes and conditions that have a cellular injury and/or stress component. They would provide capabilities that are currently either highly limited or entirely lacking.

描述（由申请人提供）：许多神经系统疾病和病理包括或假设包括细胞损伤和/或压力，作为该机制的一部分。实验方法检查这些机制，甚至确定它们在某些条件下是否确实在发挥作用，通常受到限制。它们包括对已知胁迫响应基因/分子的验尸后分析，体内药理学操纵通常是全身性的，或功能评估/操纵，通常无法将压力与非压力神经元分开。理想情况下，有可能根据细胞确定先验混合种群中哪些神经元表现出细胞压力反应。此外，由于某些压力反应或至少有压力反应的某些组成部分，即使总体压力反应可能会对细胞产生累积影响，也只能暂时表达，因此具有先验性指示神经元在过去某个时候表现出压力反应的先验指示非常有用。能够选择性地评估应激神经元的细胞和细胞间功能也将非常有用。在这些目的上，我们建议生成新的功能性转基因小鼠系，以结合这些特征。记者将由激活转录因子3（ATF3）的启动子区域（ATF3）驱动，这是一种参与多种细胞应激反应的“集线器”蛋白。为了保留天然ATF3等位基因的功能，这是某些细胞过程所必需的。带有ATF3基因座的BAC转基因将促进融合到荧光蛋白的光门控离子通道旋转2（CHR2）的产生。一个模型将以模拟方式产生记者（即反映天然ATF3表达）。第二个模型将使用诱导的Cre-cobinase系统永久“切换”报告基因生产，从而允许在任何以后的时间识别先前表示压力反应的神经元。这些功能性重复蛋白模型将允许离线解剖学评估，FACS或激光捕获分离，以前应激的神经元的命运追踪以及体内或体外功能评估，专门针对压力神经元（即表达CHR2的神经元）。公共卫生相关性：该项目旨在生成研究工具，尤其是2种新的转基因小鼠行，这些小鼠将产生功能性和解剖学记者，以响应细胞压力。这些线将显着增强基础科学方法，以理解具有细胞损伤和/或压力成分的神经系统过程和状况。他们将提供目前高度有限或完全缺乏的功能。