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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：
8464294
负责人：
TSONWIN HAI
金额：
$ 18.14万
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 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8464294
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).

描述（由申请人提供）：许多神经系统疾病和病理学包括或假设包括细胞损伤和/或应激作为机制的一部分。检查这些机制，甚至确定它们是否确实在某些条件下发挥作用的实验方法通常是有限的。它们包括对已知应激反应基因/分子的尸检分析、通常是系统性的体内药理学操作，或通常无法将应激神经元与非应激神经元分开的功能评估/操作。理想情况下，可以在逐个细胞的基础上先验地确定，混合群体中的神经元表现出细胞应激反应。此外，由于一些应激反应，或至少应激反应的某些组成部分，即使整体应激反应可能对细胞产生累积效应，也只是短暂表达，因此预先指示哪些神经元在过去的某个时刻表现出应激反应将非常有用。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。为此，我们建议产生新的功能报告基因转基因小鼠品系，其中将包含这些特征。报告基因将由激活转录因子 3 (ATF3) 的启动子区域驱动，ATF3 是一种参与多种细胞应激反应的“枢纽”蛋白。小鼠的产生将通过 BAC 转基因进行，以保留天然 ATF3 等位基因的功能，这对于某些细胞过程是必需的。带有 ATF3 基因座的 BAC 转基因将驱动与荧光蛋白融合的光门控离子通道通道视紫红质-2 (ChR2) 的产生。一种模型将以模拟方式生成记者（即，反映本地 ATF3 表达）。第二种模型将使用诱导型 Cre 重组酶系统来永久“开启”报告基因的产生，从而允许在以后的任何时间识别先前表达应激反应的神经元。这些功能报告模型将允许离线解剖评估、FACS 或激光捕获分离、先前应激神经元的命运追踪，以及特别针对应激神经元（即表达 ChR2 的神经元）的体内或体外功能评估。