GENETIC MODIFICATION OF THE CENTRAL NERVOUS SYSTEM

中枢神经系统(CNS)由许多通路和 原子核。中枢神经系统中大多数解剖学上可定义的结构是由 许多不同的单元格类别，每个单元格类别都被推定为 对整个结构功能的具体贡献。一个 理解结构作用的基本问题 多个相互联系的细胞群体是很少被 有可能，使用传统的神经生物学方法，明确地 定义单个神经元群体在复杂结构中的作用。 在本申请中，我们建议使用最近开发的技术， 基因消融，实现了一类选择消除 从中枢神经系统提取神经元，并随后研究该细胞的作用 CNS结构和功能中的类。我们特别建议： 从视网膜中清除已识别的细胞类别。世界银行的目标是 建议的实验是：1)识别视网膜细胞类别的特异性 从视网膜消减文库中分离基因，2)分离启动子 识别基因的区域，3)通过融合构建融合基因 已鉴定基因对白喉减毒毒素的启动子 基因，4)使用该融合基因创造转基因小鼠和5)使用 这些转基因小鼠在一系列解剖学和神经生理学方面 旨在描述消融对心脏的影响的评估 视网膜的结构和功能。在我们的初步实验中， 我们已经成功地清除了视网膜上的杆状感光器， 从而证明这是一种可行的方法，对于选择性的 从中枢神经系统中去除特定的细胞群。我们选择了 视网膜作为基因消融应用的模型系统 原因有很多。首先，它是一个层状结构，有体细胞 主要的、公认的细胞类别及其过程，存在于特定的 片层。其次，视网膜神经节细胞是唯一的输出 视网膜神经元及其反应反映了 视网膜外周成分活动较多。 因此，这些细胞提供了一种可获得的体内实验方法。 是一种消融。最后，我们的实验室拥有相当多的专业知识 对视网膜的解剖学和生理学分析。而当 本申请中描述的实验将在 视网膜中的神经元，我们认为必须强调的是 我们选择的方法将同样强大地定义 具有独特遗传物质的任何一组细胞的功能 被指认出来。

The central nervous system (CNS) is comprised of numerous pathways and nuclei. Most anatomically definable structures in the CNS are composed of many different cell classes, each of which is presumed to make a specific contribution to the function of the entire structure. A fundamental problem in understanding the role of structures with multiple, interconnected cell populations is that it has seldom been possible, using traditional neurobiological methods, to unequivocally define the role of single populations of neurons in a complex structure. In this application, we propose to use a recently developed technique, genetic ablation, to achieve the selection elimination of a class of neurons from the CNS and subsequently, to study the role of that cell class in CNS structure and function. In particular, we propose to eliminate an identified cell class from the retina. The goals of the proposed experiments are: 1) to identify retinal cell-class specific genes from subtracted retinal cDNA libraries, 2) to isolate the promoter regions of the identified genes, 3) to construct fusion genes by fusing the promoter of the identified gene to an attenuated diphtheria toxin gene, 4) to use this fusion gene to create transgenic mice and 5) to use these transgenic mice in a series of anatomical and neurophysiological assessments designed to characterize the effects of the ablation on the structure and function of the retina. In our preliminary experiments, we have successfully eliminated the rod photoreceptors from the retina, thereby demonstrating that this is a viable approach for the selective ablation of a specific cell population from the CNS. We have chosen the retina as our model system for the application of genetic ablation for a number of reasons. first, it is a laminar structure, with the somata of major, recognized cell classes, and their processes, lying in specific laminae. Second, retinal ganglion cells represent the sole output neurons of the retina and their responses reflect the integration of the activity of the more peripheral retinal elements. These cells, therefore, offer an accessible in vivo assay for the effects of an ablation. Finally, our laboratory has considerable expertise in the analysis of the retina, both anatomically and physiologically. While the experiments described in this application will be performed on neurons in the retina, we fell it is important to emphasize that the approach that we have chosen will be equally powerful in defining the function of any population of cells for which unique genetic material can be identified.