Novel transgenic mice for tracing and electrophysiology of stressed neurons

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，甚至确定它们是否在某些条件下确实起作用，通常是有限的。它们包括已知应激反应基因/分子的死后分析，体内药理学操作（通常是全身性的）或功能评估/操作（通常不能将应激神经元与非应激神经元分开）。理想情况下，有可能在逐个细胞的基础上先验地确定， 混合群体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应激反应的至少一些组分仅瞬时表达，即使总体应激反应可能对细胞具有累积效应，因此具有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。能够选择性地评估应激神经元的细胞和细胞间功能也将是非常有用的。为此，我们建议产生新的功能报告转基因小鼠品系，将这些特点。报告基因将由转录激活因子3（ATF 3）的启动子区驱动，ATF 3是一种参与各种细胞应激反应的“中枢”蛋白。将通过BAC转基因产生小鼠，以保留某些细胞过程所必需的天然ATF 3等位基因的功能。具有ATF 3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（ChR 2）的产生。一种模型将以模拟方式产生报告子（即，以反映天然ATF 3表达）。第二种模型将使用可诱导的Cre重组酶系统来永久地“开启”报告基因的产生，从而允许在任何以后的时间， 之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、先前应激的神经元的命运追踪以及特别是应激神经元的体内或体外功能评估（即，表达ChR 2的那些）。

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).