Generation of Brain Subregion-Restricted Conditional Tra

大脑的巨大复杂性来自于数百种神经元细胞类型和它们之间广泛的突触连接。传统上，通过各种脑损伤技术，如抽吸、电损伤或药物给药，促进了对脑亚区局部功能的研究。然而，这些程序通常影响比预期更多的大脑区域，可能导致投射区域的功能受损。为了克服这些局限性，新的条件转基因技术已经通过基因工程的发展而发生了革命性的变化，基因工程理想地在体内某个脑亚区的特定细胞类型中打开和关闭基因表达。例如，P1噬菌体的Cre重组酶已被证明对于成年大脑的有丝分裂后神经元细胞中的条件转基因操作是非常宝贵的。自2003年3月以来，我们已经启动了一个项目，创造了各种脑亚区或细胞类型限制的条件转基因小鼠，我们将推动我们了解大脑亚区在高级认知功能中的重要性，如学习和记忆，情绪状态，包括焦虑和恐惧，注意力和意识。第一年的目标是创建各种亚区限制性Cre重组酶转基因系，因为预计我们将能够通过将其与NMDA受体亚基1（NR1）的同源floxed小鼠品系杂交，将N-甲基-D-谷氨酸（NMDA）受体（突触可塑性的关键兴奋性氨基酸受体）敲除靶向几个脑区。本课题的关键问题是选择基因启动子，决定转基因表达的细胞类型或脑区特异性。由于携带海马CA3限制性表达启动子的BAC（细菌人工染色体）克隆已经从Kazu Nakazawa的先前研究中获得，因此CA3限制性转基因项目现在作为其自己的项目正在进行中。基因蛋白质合成敲除小鼠项目也已分离为一个独立项目，如单独描述的。从文献和我们的原位杂交组织化学，我们已经进一步确定了一些BAC克隆携带的基因表达的启动子主要在海马CA1区，杏仁核，内嗅皮层，前额叶皮层，前脑中间神经元和丘脑核，分别。虽然以前没有鉴定指导每种基因产物靶向表达的遗传增强子/启动子的报道，但现在可以在Pub-Med网站上获得每种BAC克隆的整个DNA的核苷酸序列。从这些序列的广泛的基于计算机的分析，我们估计了推定的DNA片段携带这样的区域，并通过使用脉冲场凝胶电泳（PAGE）凝胶纯化每个BAC克隆。我们还纯化了带有核定位信号的Cre重组酶cDNA的DNA片段。然后，在转基因核心设施（Dr.JamesPickel）的帮助下，我们将Cre cDNA和BAC片段的DNA片段共注射到小鼠受精卵中，以获得转基因品系。一旦携带Cre-和BAC-DNA的双阳性系被建立为来自其后代的转基因系，我们将它们与Rosa26报告系杂交，其中Cre重组酶的表达通过X-gal染色功能性地可视化。大多数实验室成员都参与了这个项目; Yuichi Hirata博士参与了前额叶皮层项目，现在由Kimberly Christian博士负责。她目前也在进行内嗅皮层项目。姜志宏博士主要参与海马CA1区项目，同时指导了大部分BAC片段的纯化。Juan Belforte博士是NIAAA支持的访问学者，他正在研究几个含有推定的NAcc遗传启动子的BAC克隆。凯瑟琳·克雷文斯参与了这些项目中的许多基因分型？线Kazu Nakazawa参与了杏仁核和中间神经元项目，并监督其他实验室成员。目前，我们的实验室正在维护和分析至少几个Cre/BAC双阳性转基因株系，分别用于CA1，杏仁核，内嗅皮层，前额叶皮层和前脑中间神经元的项目。由于Rosa26杂交后的F1分析在过去几个月才开始，我们还没有发现任何好的区域限制Cre系。尽管如此，我们预计这些细胞系中的一些将在不久的将来提供最先进的细胞类型限制的Cre重组酶在脑中的过表达。一旦建立了这些细胞系，我们将进一步缩小该项目的范围，将NMDA受体敲除靶向特定的细胞类型，并研究NMDA受体区域限制性敲除的行为和生理后果，以了解最严重的神经精神疾病，如双相情感障碍和精神分裂症。

The enormous complexity of the brain is derived from hundreds of neuronal cell types and extensive synaptic connections between them. Studies of the localized function of the brain-subregions have traditionally been facilitated by the various brain lesion techniques such as aspiration, electrical lesion, or pharmacological administrations. However, these procedures often influenced more brain areas than expected, potentially resulting in the impaired function of the projection area as well. To overcome these limitations, new conditional transgenic technologies have been revolutionized by the development of genetic engineering that ideally switches gene expression on and off in a particular cell-type of a certain brain subregion in vivo. For example, Cre recombinase of the P1 bacteriophage has proven invaluable for conditional transgenic manipulation in post-mitotic neuronal cells of the adult brain. Since March 2003, we have initiated a project to create a variety of brain-subregion or cell-type restricted conditional transgenic mice, which we will move us toward understanding of the significance of brain subregions in higher cognitive functions, such as learning and memory, emotional state including anxiety and fear, attention, and awareness. The goal of the first year was to create various subregion-restricted Cre-recombinase transgenic lines, since it is expected that we will be able to target the knockout of N-methyl-D-asparate (NMDA) receptor, a critical excitatory amino acid receptor for synaptic plasticity, into several brain areas by crossing them with a homozygously-floxed mouse strain of NMDA receptor subunit 1 (NR1). The key issue of this project was the choice of genetic promoter which determines the cell type or brain subarea specificity of transgene expression. Since a BAC (bacterial artificial chromosome) clone carrying a promoter for hippocampal CA3 restricted expression was already in our hand from the previous study of Kazu Nakazawa, the CA3-restricted transgenic project is now underway as its own project. The project of genetic protein synthesis knockdown mice has also separated to an independent project as described separately. From the literatures and by our in situ hybridization histochemistry, we have further identified some BAC clones that carry promoters for the gene expression predominantly in the hippocampal CA1, amygdala, entorhinal cortex, prefrontal cortex, forebrain interneurons and nucleus accumbens, respectively. While there were no previous reports identifying the genetic enhancer/promoter which direct the targeted expression of each gene product, the nucleotide sequence of the whole DNA of each BAC clone is now available on the Pub-Med web site. From the extensive computer-based analysis of these sequences, we estimated the putative DNA fragments carrying such regions and purified them by using the pulse-field gel electrophoresis (PAGE) gels for each BAC clone. We also purified a DNA fragment carrying Cre-recombinase cDNA with a nuclear localization signal. Then, with the great help of the Transgenic Core Facility (Dr. James Pickel), we co-injected the DNA fragments of Cre cDNA and BAC fragment, into mouse fertilized eggs to generate transgenic lines. Once the double positive lines carrying both Cre- and BAC-DNA are established as a transgenic line from their offspring, we crossed them with a Rosa26 reporter line, in which the expression of Cre recombinase is functionally visualized by X-gal staining. Most of the lab members have participated in this project; Dr. Yuichi Hirata was involved in the prefrontal cortex project, which is now followed by Dr. Kimberly Christian. She is currently working on entorhinal cortex project as well. Dr. Zhihong Jiang is mainly involved in hippocampal CA1 project, while she supervised the purification of most of BAC fragments. Dr. Juan Belforte, a visiting fellow supported by NIAAA, is workinng on several BAC clonees which contain putative NAcc genetic promoter. Catherine Cravens is engaged in the genotyping many of these projects? lines. Kazu Nakazawa is involved in both amygdala and interneuron projects, as well as supervising other lab members. Currently, our lab is maintaining and analyzing at least a few transgenic lines of Cre/BAC double-positive for the project of CA1, amygdala, entorhinal cortex, prefrontal cortex, and forebrain interneurons, respectively. Since the F1 analysis following Rosa26 crossing has just started in the past few months, we do not find any of good region-restricted Cre lines yet. Nevertheless, we expect some of these lines will provide a state-of-art cell type-restricted over-expression of Cre recombinase in the brain in the near future. Once these lines are established, we will further narrow down this project to target the NMDA receptor knockout to particular cell types and investigate the behavioral and physiological consequence of region-restricted knockout of NMDA receptors to our understanding of the most serious neuropsychiatric disorders, such as bipolar disorders and schizophrenia.