Novel transgenic mice for tracing and electrophysiology of stressed neurons

描述(由申请人提供)：许多神经疾病和病理包括或假设包括细胞损伤和/或应激作为机制的一部分。检验这些机制的实验方法，甚至确定它们是否确实在特定条件下发挥作用的实验方法，通常都是有限的。它们包括已知的应激反应基因/分子的死后分析，通常是系统性的体内药物操作，或者往往无法将应激和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确定先验结果， 混合种群中的哪些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成分，即使总体应激反应可能对细胞产生累积影响，但仅是瞬时表达的，因此对哪些神经元在过去的某个时间点表现出应激反应的先验指示将是非常有用的。能够有选择地评估应激神经元的细胞和细胞间功能也是非常有用的。为此，我们建议建立新的功能报告转基因小鼠品系，该品系将包含这些特征。这些记者将由激活转录因子3(ATF3)的启动子区域驱动，ATF3是一种参与各种细胞应激反应的“中枢”蛋白。为了保存某些细胞过程所必需的天然ATF3等位基因的功能，将通过BAC转基因来产生小鼠。带有ATF3基因的BAC转基因将驱动光门控离子通道-视紫红质-2(ChR2)融合到荧光蛋白的生产。一个模型将以模拟方式制作记者(即，反映原生ATF3表达)。第二个模型将使用可诱导的Cre重组酶系统永久地“开启”报告生产，从而允许在任何以后的时间识别 以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学评估、FAC或激光捕获分离、先前应激神经元的命运追踪以及体内或体外特定应激神经元(即表达ChR2的神经元)的功能评估。

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).