Generation of Brain Subregion-Restricted Conditional Tra

大脑的巨大复杂性源于数百种神经细胞类型和它们之间广泛的突触联系。脑区局部功能的研究传统上是通过各种脑损伤技术来促进的，如抽吸、电损伤或药物给药。然而，这些程序往往比预期影响更多的大脑区域，潜在地导致投影区功能受损。为了克服这些限制，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，这种基因工程理想地在体内特定脑区的特定细胞类型中开启和关闭基因表达。例如，P1噬菌体的Cre重组酶已被证明对于成人大脑有丝分裂后神经细胞的条件转基因操作是非常有价值的。自2003年3月以来，我们已经启动了一个项目来创造各种脑区或细胞型限制性条件转基因小鼠，我们将帮助我们理解大脑亚区在高级认知功能中的重要性，如学习和记忆，包括焦虑和恐惧的情绪状态，注意力和意识。第一年的目标是创建各种区域限制性Cre重组酶转基因株系，因为预计我们将能够通过将N-甲基-D-天冬氨酸(NMDA)受体(一种对突触可塑性起关键作用的兴奋性氨基酸受体)与NMDA受体亚单位1(NR1)纯合子漂浮的小鼠品系杂交，将其敲除到几个脑区。这个项目的关键问题是基因启动子的选择，它决定了转基因表达的细胞类型或脑区特异性。由于中泽一郎之前的研究已经获得了携带海马区CA3限制性表达启动子的BAC(细菌人工染色体)克隆，因此CA3限制性转基因项目目前正在作为自己的项目进行。基因蛋白合成击倒小鼠的项目也已分离为单独描述的一个独立项目。根据文献和我们的原位杂交组织化学方法，我们进一步鉴定了一些携带该基因表达启动子的BAC克隆，它们分别在海马CA1、杏仁核、内嗅皮层、前额叶皮质、前脑中间神经元和伏隔核中。虽然以前没有报告确定指导每个基因产物的靶向表达的遗传增强子/启动子，但每个BAC克隆的整个DNA的核苷酸序列现在可以在Pub-Med网站上找到。通过对这些序列的大量计算机分析，我们估计了可能携带这些区域的DNA片段，并使用每个BAC克隆的脉冲场凝胶电泳(PAGE)凝胶对它们进行了纯化。我们还纯化了携带带有核定位信号的Cre重组酶基因的DNA片段。然后，在转基因核心设备(Dr.James Pickel)的帮助下，我们将Cre基因的DNA片段和BAC片段共同注射到小鼠受精卵中，获得了转基因系。一旦同时携带Cre-DNA和BAC-DNA的双阳性品系从它们的后代中建立为转基因系，我们将它们与一个rosa26报告系杂交，其中Cre重组酶的表达通过X-Gal染色进行功能可视化。大多数实验室成员都参与了这个项目；平田雄一博士参与了前额叶皮质项目，现在金伯利·克里斯蒂安博士参与了该项目。她目前也在进行内嗅皮层项目。姜志红博士主要参与了海马区CA1区的研究工作，并监督了大部分BAC片段的纯化工作。Juan Belforte博士是NIAAA支持的访问研究员，他正在研究几个BAC克隆，这些克隆包含假定的NAcc基因启动子。凯瑟琳·克雷文斯参与了许多这样的项目的基因分型？台词。中泽一郎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，我们实验室正在维护和分析至少几个Cre/BAC转基因系，分别用于CA1、杏仁核、内嗅皮层、前额叶皮质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刚刚开始，我们还没有发现任何好的区域限制的CRE品系。然而，我们预计其中一些株系将在不久的将来提供一种最先进的细胞类型限制性的Cre重组酶在大脑中的过度表达。一旦建立了这些线，我们将进一步缩小这个项目的范围，将NMDA受体基因敲除的目标锁定到特定的细胞类型，并调查区域限制性NMDA受体基因敲除的行为和生理后果，以帮助我们了解最严重的神经精神障碍，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.