Generation of Brain Subregion-Restricted Conditional Tra

大脑分区限制条件训练的生成

• 批准号: 6982743
• 负责人: Kazutoshi Nakazawa
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6982743
• 关键词: amygdala; animal genetic material tag; artificial chromosomes; biotechnology; brain mapping; cell type; entorhinal cortex; expression cloning; gene expression; genetic promoter element; genetic regulation; genetically modified animals; hippocampus; in situ hybridization; laboratory mouse; neurogenesis; nucleus accumbens; prefrontal lobe /cortex; protein biosynthesis; recombinant DNA; recombinase; tegmentum; amygdala; animal genetic material tag; artificial chromosomes; biotechnology; brain mapping; cell type; entorhinal cortex; expression cloning; gene expression; genetic promoter element; genetic regulation; genetically modified animals; hippocampus; in situ hybridization; laboratory mouse; neurogenesis; nucleus accumbens; prefrontal lobe /cortex; protein biosynthesis; recombinant DNA; recombinase; tegmentum

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.

大脑的巨大复杂性来自数百种神经元细胞类型和它们之间的广泛突触连接。传统上，诸如吸入，电病变或药理学施用等各种脑病变技术促进了有关大脑区域局部功能的研究。但是，这些程序通常会影响大脑面积超过预期的大脑区域，这也可能导致投影区域的功能受损。为了克服这些局限性，通过基因工程的发展发展了新的条件转基因技术，理想地在体内特定细胞型中开关和关闭基因表达。例如，P1噬菌体的CRE重组酶对成人大脑的有条件转基因操纵而言已证明是无价的。自2003年3月以来，我们启动了一个项目，以创建各种脑部或细胞类型的有条件转基因小鼠，我们将朝着理解大脑子区域在较高认知功能中的重要性，例如学习和记忆，等情绪状态，包括焦虑和恐惧，注意力，注意力，注意力和认识。第一年的目的是创建各种限制的子区域CRE成分酶转基因线，因为可以通过将N-Methyl-D-D-A隔离（NMDA）受体的敲除敲除n--甲基-D-DA的受体（一种关键的兴奋性氨基酸受体），这是一种突触可变性的关键兴奋性氨基酸受体，将其与均质型flosy-floblo fellozy-Flobomey-Fbore-unny nMda cromenter crontapticotition nmda nmda croment rymozy nmda nmda。该项目的关键问题是选择遗传启动子，该基因启动子决定了转基因表达的细胞类型或脑部亚区域特异性。由于带有海马CA3启动子的BAC（细菌人造染色体）克隆限制性表达的启动子已经在先前的Nakazawa研究中，因此CA3限制的转基因项目现在正在作为其自己的项目。遗传蛋白质合成敲低小鼠的项目也已分别分别为一个独立的项目。从文献和我们的原位杂交组织化学中，我们进一步鉴定了一些BAC克隆，这些克隆主要在海马CA1，杏仁核，内hinal骨皮质，前额叶皮质，前额叶皮质，前脑皮层，前脑间间和静脉内神经元和核子和核酸核酸杆菌中携带基因表达的启动子。尽管没有以前的报道识别指导每个基因产物的靶向表达的遗传增强子/启动子，但现在每个BAC克隆的整个DNA的核苷酸序列现在都可以在Pub-Med网站上获得。根据对这些序列的广泛基于计算机的分析，我们估计了携带此类区域的假定DNA片段，并通过为每个BAC克隆使用脉冲场凝胶电泳（PAGE）凝胶纯化它们。我们还纯化了带有核定位信号的CRE聚合酶cDNA的DNA片段。然后，在转基因核心设施（James Pickel博士）的大力帮助下，我们将CRE cDNA和BAC片段的DNA片段共同注入小鼠受精卵，以产生转基因线。一旦将带有CRE-和BAC-DNA的双阳性线从其后代建立为转基因线，我们就将它们与Rosa26报告基因线越过，其中Cre Rebombinase的表达通过X-Gal染色在功能上可视化。大多数实验室成员都参加了该项目。 Yuichi Hirata博士参与了前额叶皮层项目，其次是金伯利·克里斯蒂安（Kimberly Christian）博士。她目前也在从事Entorhinal Cortex项目。江江博士主要参与海马CA1项目，而她监督了大多数BAC碎片的纯化。由NIAAA支持的来访者Juan Belforte博士正在使用一些BAC克隆，其中包含推定的NACC遗传启动子。凯瑟琳·克雷文斯（Catherine Cravens）从事许多这些项目的基因分型？线。纳川（Kazu Nakazawa）参与了Amygdala和Interneuron项目，并监督其他实验室成员。目前，我们的实验室分别为CA1，杏仁核，内嗅皮层，前额叶皮层和前脑中神经元的项目维持和分析至少几条CRE/BAC双重阳性的转基因线。由于在过去几个月中，Rosa26穿越之后的F1分析刚刚开始，因此我们还没有发现任何良好的区域限制的CRE线。然而，我们预计其中一些线条将在不久的将来提供大脑中CRE重组酶的最先进的细胞类型限制过表达。一旦建立了这些线条，我们将进一步缩小该项目的范围，以将NMDA受体敲除靶向特定的细胞类型，并研究NMDA受体区域限制敲除的行为和生理后果，以了解我们对最严重的神经精神疾病的理解，例如双极性疾病和精神分裂症。