Generation and characterization of novel and highly specific neruonal subtype tra

描述(申请人提供)：转基因工具的用途，可以选择性地标记每一种特定类型的神经元，此外，选择性地使基因失活或一次操纵一种类型神经元的活动，在神经科学研究的许多不同领域具有很高的影响。目前许多人采取的方法，例如Cre驱动系的建立，通常利用在某些细胞类型中相对特异表达的基因。然而，这种“标记”基因很少给真正特定的神经元群体贴上标签。进一步将靶标特异性细化到特定区域或特定的神经元群体是非常必要的。为了实现这一点，我们建议系统地评估一些组合策略，并开发一系列转基因工具，通过两个标记基因的交叉表达来驱动高度特异的基因表达。通过挖掘我们的全基因组数据库Allen Brain Atlas，曾博士和他的同事们已经确定了不同皮质细胞类型的一大组基因标记。纳吉博士开发了一套基因打靶和等位基因替换策略，以有效地表达多个基因。艾伦研究所拥有一个高吞吐量的平台和高分辨率图像采集和数据库的能力。通过结合双方开发的精湛技术，我们将利用双重荧光ISH(DFISH)检查不同标记基因的共存，创建驱动和报告/响应小鼠品系来测试不同的交叉策略，并再次使用dFISH来系统地表征和建立小鼠整个大脑中受控基因表达的数据库。在这笔赠款中，我们将使用新大脑皮层作为建立技术的模型。一旦完成，它也可以很容易地应用于其他大脑区域。与公共健康相关：研究大脑的极端复杂性需要找到方法来梳理其组成部分。传统的转基因工具已广泛应用于神经科学研究的各个领域。我们提出的研究将为神经科学界提供两种类型的强大工具：转基因小鼠品系，可以将基因操作引导到高度特定的神经元群体，以及一个全面记录这种操作发生地点的图像数据库。这两种工具都将对广泛的神经科学界产生重大影响，从分子到系统神经科学，从发育到行为研究，从正常大脑生理研究到疾病机制研究。

DESCRIPTION (provided by applicant): The utility of transgenic tools that can selectively label each particular type of neuron, and furthermore, selectively inactivate genes or manipulate the activity of one type of neuron at a time, has high impact in many different areas of neuroscience research. Current approaches taken by many, e.g. the Cre-driver line creation, usually utilize genes relatively specifically expressed in certain cell types. However, such "marker" genes rarely label a really specific neuronal population. It is highly desirable to further refine the target specificity into a particular region or a specific population of neurons. To achieve this we propose to systematically evaluate a number of combinatorial strategies and develop a series of transgenic tools to drive highly specific gene expression through the intersection of the expression of 2 marker genes. Through mining our genome-wide database, Allen Brain Atlas, Dr. Zeng and colleagues have identified a large set of gene markers for different cortical cell types. Dr. Nagy has developed a set of gene targeting and allele replacement strategies for expressing multiple genes efficiently. Allen Institute has a high throughput ISH platform and capability of high resolution image acquisition and databasing. By combining the superb technologies developed by both parties, we will examine the colocalization of different marker genes by double fluorescent ISH (dFISH), create both driver and reporter/responder mouse lines to test different intersection strategies, and use the dFISH again to systematically characterize and database where the controlled gene expression occurs in the entire mouse brain. In this grant we will use the neocortex as a model to establish the technology. Once completed, it can be applied easily into other brain regions as well. PUBLIC HEALTH RELEVANCE: Studying the extreme complexity of the brain demands ways to tease apart its components. Traditional transgenic tools have been widely used in every area of neuroscience research. Our proposed research will provide two types of powerful tools to the neuroscience community: transgenic mouse lines that can direct genetic manipulation to highly specific neuronal populations, and an image database comprehensively documenting where such manipulation occurs. Both of these tools will have major impact on the broad neuroscience community, from molecular to systems neuroscience, from development to behavior studies, from studies of normal brain physiology to disease mechanisms.