Gene expression nervous system atlas (GENSAT)
Gene expression nervous system atlas (GENSAT)
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DOI：
10.1038/nn0504-483
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发表时间：
2004-05-01
影响因子：
25
通讯作者：
Heintz, N
中科院分区：
文献类型：
--
作者：
Heintz, N
我们对大脑形成和功能的分子机制的理解必须包括关于特定基因和蛋白质在整个发育过程中的精确分布的信息，以及识别、可视化和遗传操纵每一种主要中枢神经系统(CNS)细胞类型的能力。GENSAT项目的目的是利用各种研究的结果来确定和定位∼5,000在整个发育过程中最重要的中枢神经系统表达基因的表达，创建一个具有特征的细菌人工染色体克隆文库，为哺乳动物大脑中的每个主要细胞群体提供遗传通道，并建立一个携带荧光报告基因的细菌人工染色体转基因小鼠品系集合，以便对这些细胞进行进一步的解剖学和生理学研究。BAC中携带的大染色体片段确保了大多数基因在整合到小鼠基因组后保持可复制的表达。GENSAT项目是第一个将原位杂交和BAC转基因方法相结合的大规模努力，以创建哺乳动物大脑中基因表达的图谱，同时改善对所有类别中枢神经系统细胞的遗传获取。该项目使用两种方法来绘制基因表达图谱。首先，在T.Curran、S.Magdaleno和P.Jensen的监督下，在田纳西州孟菲斯的圣裘德儿童研究医院进行了高通量的基因表达原位杂交预筛选，以绘制出每年从发育中的和成年小鼠大脑切片上超过1000个基因的表达情况。作为预筛选的一部分，分析的基因由代表神经科学界广泛领域的咨询委员会、国家神经疾病和中风研究所(NINDS)的项目发起人以及GENSAT调查人员选择。其次，每年从筛选前数据中选择250个基因，使用加入到BAC转基因小鼠中的增强型绿色荧光蛋白(EGFP)报告基因进行高分辨率分析(在N.Heintz、MB Hatten和A.Joyner的指导下，在洛克菲勒大学)。这种两阶段的方法利用同位素原位杂交方法的敏感性、动态范围和效率来并行分析大量CNS表达的基因，同时利用BAC报告基因技术来系统分析和高分辨率可视化每种细胞类型表达感兴趣的基因。除了GENSAT团队创建的基因表达图谱外，BAC转基因小鼠还被多个实验室用于高级成像研究、通过荧光激活细胞分选分离细胞、对已定义的中枢神经系统细胞群进行电生理分析，以及其他解剖学研究。此外，GENSAT鉴定的BAC载体通过表达特定部位的重组酶、显性激活或显性负性蛋白和RNA以及其他用于改变细胞性质的分子，为特定CNS细胞类型的额外遗传学研究提供了丰富的资源。我们预计，基因表达图谱以及原位探针、BAC转基因小鼠和GENSAT产生的BAC载体将成为广泛的神经科学家的重要工具。
Our understanding of the molecular mechanisms that contribute to the formation and function of the brain must include information about the precise distributions of specific genes and proteins throughout development, and the ability to identify, visualize and genetically manipulate each of the major central nervous system (CNS) cell types. The aims of the GENSAT project are to use results from a variety of studies to identify and map the expression of∼ 5,000 of the most important CNS-expressed genes throughout development, to create a characterized library of bacterial artificial chromosome (BAC) clones that provide genetic access to each of the major cell populations in the mammalian brain, and to establish a collection of BAC transgenic mouse lines carrying fluorescent reporter genes that allow further anatomical and physiological studies of these cells. The large chromosomal segments carried in the BACs ensure that reproducible expression is retained for most genes after integration into the mouse genome. The GENSAT project is the first large-scale effort to combine in situ hybridization and BAC transgenic methods to create an atlas of gene expression in the mammalian brain, while improving genetic access to all classes of CNS cells.The project uses two approaches to gene-expression mapping. First, a high-throughput isotopic in situ hybridization prescreen for gene expression is done to map the expression of over 1,000 genes per year on sections from developing and adult mouse brain (at St. Jude Children's Research Hospital, Memphis, Tennessee under the supervision of T. Curran, S. Magdaleno and P. Jensen). The genes analyzed as part of the prescreen are chosen by advisory committees representing a broad spectrum of the neuroscience community, by project sponsors at the National Institute of Neurological Disorders and Stroke (NINDS) and by the GENSAT investigators. Second, 250 genes are chosen each year from the prescreen data to be analyzed at high resolution using enhanced green fluorescent protein (EGFP) reporter genes incorporated into BAC transgenic mice (at The Rockefeller University under the direction of N. Heintz, MB Hatten and A. Joyner). This two-stage approach takes advantage of the sensitivity, dynamic range and efficiency of isotopic in situ hybridization methodology to allow parallel analysis of large numbers of CNS-expressed genes, while exploiting BAC reporter gene technology to allow systematic analysis and high-resolution visualization of each cell type expressing a gene of interest. In addition to the atlas of gene expression created by the GENSAT team, the BAC transgenic mice are being used in a number of laboratories for advanced imaging studies, cell isolation by fluorescence-activated cell sorting, electrophysiological analysis of defined CNS cell populations, and additional anatomical studies. Furthermore, the BAC vectors identified by GENSAT provide a rich resource for additional genetic studies of specific CNS cell types through expression of site-specific recombinases, dominant-activating or dominant-negative proteins and RNAs, and other molecules created to alter the properties of cells. We anticipate that the gene expression atlas, as well as the in situ probes, the BAC transgenic mice and the BAC vectors generated by GENSAT, will be important tools for a broad range of neuroscientists.