Generation of Brain Subregion-Restricted Conditional

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。传统上，对脑亚区局部功能的研究是通过各种脑损伤技术来促进的，例如吸入性损伤、电损伤或鹅膏酸损伤。然而，这些手术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，如钠通道阻滞剂TTX和GABA激动剂Muscimol，也被用来阐明它们的局部功能。这种方法具有更强的分子选择性和可逆的时间可控性。然而，药物进入脑区并不区分特定的细胞类型，往往会导致细胞损伤和毒性。为了克服这些限制，遗传工程的发展使一种新的条件转基因技术发生了革命性的变化，它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。四环素反应系统也被证明在大脑中以一种可逆的方式有用。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，我们发起了一个项目，创造各种脑区或细胞类型的限制性条件转基因小鼠。由于任何特定的大脑功能都会受到许多脑区的影响，我们关注的是几个脑区，如海马CA1/CA3、杏仁核、内嗅皮层、前额叶皮质、伏隔核和腹侧被盖区。该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。其中，我们之前的研究中发现了一个携带海马区CA3限制性表达启动子的细菌人工染色体克隆。我们进一步鉴定了大多数携带基因表达启动子的BAC克隆，分别位于上述脑区。目前，我和我的同事正在构建转基因小鼠的DNA结构，其中Cre重组酶将主要在上述脑亚区表达。与此同时，通过结合Cre/loxP系统，我们正试图以细胞类型特异性的方式建立蛋白质合成的基因敲除系统。在几年内，我们有望建立各种脑区受限的基因操作系统，有望为在系统水平上理解每个脑区的功能提供非常有价值的动物工具。我们还希望这项研究能带来对我们理解最严重的神经精神障碍，如双相情感障碍和精神分裂症至关重要的基础性发现。

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.