MICROARRAY ANALYSIS OF SINGLE RETINAL GANGLION CELLS

单个视网膜神经节细胞的微阵列分析

• 批准号: 6287743
• 负责人: JOSHUA R SANES
• 金额: $ 23.16万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-27 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6287743
• 关键词: biotechnology; cell type; complementary DNA; complementary RNA; developmental genetics; developmental neurobiology; genetic markers; genetically modified animals; green fluorescent proteins; in situ hybridization; laboratory mouse; microarray technology; neurogenetics; retinal ganglion

DESCRIPTION (Applicant's abstract): Retinal ganglion cells (RGCs) process visual information in the eye and transmit it to the brain. All RGCs share key features, but distinct subtypes have been identified that differ in morphology, biophysical properties, visual responsiveness, synaptic inputs and synaptic targets. These RGC subtypes have been studied intensively with anatomical and physiological methods, both because they are critical determinants of visual perception and because they provide an accessible system for addressing general issues of neuronal diversification and categorization. Unfortunately, however, few if any molecular markers are known that distinguish functionally relevant mammalian RGC subclasses, so it has been impossible to answer basic questions about how many subtypes really exist, when they arise, how they acquire their definitive identities, or what molecules underlie their subtype-specific properties. Here, we propose to identify molecular markers for two pairs of RGC subsets that have already been defined structurally and physiologically, and are of clear functional importance: alpha- vs. beta-like (which differ in soma size, dendritic spread and briskness of response) and ON vs. OFF (which differ in inputs, outputs, dendritic stratification, and responsiveness to onset vs. termination of light). To this end, we have already generated and characterized transgenic mice in which the Green Fluorescent Protein (GFP) labels RGCs of multiple types in their entirely, allowing us to identify and isolate them. We will prepare and characterize cDNA from individual transgene-positive RGCs, using methods we have recently used successfully to characterize chick RGCs. The probes will then be hybridized to commercially available microarrays (Affymetrix GeneChips), allowing us to assess expression of about 27,000 genes in cells of each of four classes (alpha-ON, alpha-OFF, beta-like-ON, beta-like-OFF). The output will be analyzed to identify genes expressed by defined RGC subsets or that define novel subsets. Finally, candidate markers will be validated by in situ hybridization. We believe these experiments will (a) provide markers for developmental studies, (b) uncover genes that themselves play roles in subtype diversification or function, and (c) contribute to development of generally applicable methods for gene expression profiling from single cells.

描述(申请人摘要)：视网膜神经节细胞(RGC)突起 眼睛中的视觉信息并将其传输到大脑。所有RGC共享密钥 特征，但已确定在形态上不同的不同亚型， 生物物理特性、视觉反应、突触输入和突触 目标。对这些RGC亚型进行了深入的解剖学和 生理学方法，因为它们都是视觉的关键决定因素 因为它们提供了一个可访问的系统来解决一般问题 神经元多样化和分类的问题。然而，不幸的是， 已知的区分功能相关的分子标记即使有也很少 哺乳动物RGC亚类，因此不可能回答基本问题 关于到底有多少子类型，当它们出现时，它们如何获得它们的 确定的身份，或其特定于亚型的分子 属性。在这里，我们建议寻找两对RGC的分子标记 已经在结构上和生理上定义的子集，以及 具有明显的功能重要性：阿尔法和贝塔(在胞体上有所不同 大小、树突扩散和响应速度)和开与关(这两者不同 在输入、输出、树枝状分层和对发病的反应性与 光的终止)。为此，我们已经生成并表征了 绿色荧光蛋白(GFP)标记RGC的转基因小鼠 它们完全具有多种类型，使我们能够识别和分离它们。我们 将从单个转基因阳性视网膜节细胞中制备和鉴定cDNAs， 使用我们最近成功使用的方法来表征雏鸡RGC。 然后，这些探针将与商业上可获得的微阵列杂交 (Affymetrix GeneChips)，使我们能够评估大约27,000个基因的表达 在四个类别(α-on、α-off、beta-like-on， Beta-Like-Off)将对输出进行分析，以确定由 定义的RGC子集或定义新的子集。最后，候选标记 将通过原位杂交进行验证。我们相信这些实验将 (A)为发育研究提供标记；(B)发现 本身在亚型多样化或功能方面发挥作用，以及(C) 有助于开发普遍适用的基因表达方法 从单个细胞进行分析。