Generation of Brain Subregion-Restricted Conditional

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、电损伤或鹅膏蕈氨酸损伤，促进了对脑亚区局部功能的研究。然而，这些程序往往会导致投射轴突的神经元变性，也可能导致投射区域的功能受损。药物干预，如TTX，钠通道阻滞剂，蝇蕈醇，GABA激动剂，到脑亚区的输液，也被用来阐明其定位功能。这种方法具有更高的分子选择性，并且以可逆的方式在时间上可控。然而，给药到脑亚区并不能区分特定的细胞类型，往往会导致细胞损伤和毒性。为了克服这些局限性，一种新的条件转基因技术已经通过基因工程的发展而革命性地发展，该基因工程理想地在体内某些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。四环素反应系统也已显示以可逆方式在脑中有用。为了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，我们启动了一个项目，以创建各种脑亚区或细胞类型限制的条件转基因小鼠。由于任何特定的大脑功能都受到许多大脑亚区的影响，我们将重点放在几个大脑亚区，如海马CA 1/CA 3，杏仁核，内嗅皮层，前额叶皮层，丘脑核和腹侧被盖区。本课题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。其中，我们在前期的研究中获得了一个携带海马CA 3限制性表达启动子的BAC克隆。我们进一步鉴定了大部分BAC克隆，它们携带的基因启动子主要在上述脑亚区表达。目前，我和我的同事正在构建用于产生转基因小鼠的DNA构建体，其中Cre重组酶将主要在上述脑亚区中表达。与此同时，通过结合Cre/loxP系统，我们试图建立一个以细胞类型特异性方式进行蛋白质合成的遗传敲低系统。在未来几年内，我们有望建立多种脑亚区限制性基因操作系统，为在系统水平上了解各脑亚区的功能提供非常有价值的动物工具。我们也希望这项研究能带来一些重要的发现，这些发现对我们理解最严重的神经精神疾病，如双相情感障碍和精神分裂症至关重要。

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 or ibotenic acid lesions. However, these procedures often caused neuronal degeneration of the projecting axons, potentially resulting in the impaired function of the projection area as well. Pharmacological intervention, such as infusion of TTX, sodium channel blocker, and muscimol, GABA agonist, into brain subregions, has also been utilized to elucidate their localized functions. This approach is more molecule selective and temporally controllable in a reversible manner. However, the drug administration into the brain subregion does not distinguish specific cell types and often lead to cellular damage and toxicity. To overcome these limitations, a new conditional transgenic technology has been revolutionized by the development of genetic engineering that ideally switches gene expression on and off in a particular cell-type of 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. Tetracycline responsive system has also been shown to be useful in the brain in a reversible manner. 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, we initiated a project to create a variety of brain-subregion or cell-type restricted conditional transgenic mice. Since any particular brain functions are affected by many brain subareas, we are focusing onto several brain subregions, such as hippocampal CA1/CA3, amygdala, entorhinal cortex, prefrontal cortex, nucleus accumbens and the ventral tegmental area. The key issue of this project is the choice of genetic promoter which determines the cell type or brain subarea specificity of transgene expression. Among them, a BAC (bacterial artificial chromosome) clone carrying a promoter for hippocampal CA3 restricted expression is in our hand from our previous study. We have further identified most of BAC clones that carries promoter for the gene expression predominantly in the above brain subregion, respectively. Currently, my colleagues and I are making DNA constructs for generation of transgenic mice in which Cre recombinase will be predominantly expressed in the above brain subregions. Concurrent with this effort, by combining Cre/loxP system, we are trying to establish a genetic knockdown system of protein synthesis in a cell-type specific manner. In a few years, we expect to establish a variety of brain subregion restricted genetic manipulation system that hopefully provides incredibly valuable animal tools for understanding of the function of each brain subregion at a system level. We also hope that this study leads to make fundamental discoveries critical to our understanding of the most serious neuropsychiatric disorders, such as bipolar disorders and schizophrenia.