Generation Brain Subregion-Conditional Transgenic Mice

一代脑亚区条件转基因小鼠

• 批准号: 7137856
• 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/7137856
• 关键词: NMDA receptors; artificial chromosomes; behavioral genetics; biotechnology; brain mapping; diphtheria toxin; dopamine; epilepsy; gene expression; genetic manipulation; genetic promoter element; genetically modified animals; hippocampus; histochemistry /cytochemistry; in situ hybridization; interneurons; laboratory mouse; mossy fiber; neurogenetics; nucleus accumbens; potassium channel; prefrontal lobe /cortex; protein biosynthesis; recombinase; transfection /expression vector

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 recently 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. Our goal this year was to create various subregion-restricted Cre-recombinase transgenic lines, using a BAC (Bacterial Artificial Chromosome) clone carrying a promoter for directing the expression in particular brain areas. From the literature and by our in situ hybridization histochemistry, we identified some BAC clones that carry promoters for the gene expression predominantly in the hippocampal CA1 (namely BAC7), amygdala (BAC1), entorhinal cortex (BAC5), prefrontal cortex (BAC4), forebrain interneurons (BAC6), ventral tegmental area (BAC8) and nucleus accumbens (BAC3 and BAC10), respectively. By September 2004, with the help of the Transgenic Core Facility (Dr. James Pickel), we had finished co-injecting the DNA fragments of HSP70 minimal promoter-Cre cDNA and DNA fragment for each BAC clone into mouse fertilized eggs to generate doubly-integrated transgenic lines. Since then we have spent much time on genotyping and analyzing the Cre recombination pattern by crossing with Rosa26 reporter LacZ line. However, fewer Cre/BAC-double transgenic lines across BAC clones were generated than expected. Out of 106 Cre positive transgenic lines we obtained from the Core Facility, only 22 lines were also BAC positive. The reason for the low yield of double transgenic mice may be due to the difficulty in preparing the same copy number of DNA fragments of such different lengths. While we are still analyzing 4 of these 22 lines, we did not obtain any ideal lines that mimic the Cre recombinase expression to the endogenous expression of each BAC gene from them. [We also analyzed over 50 Cre-positive (BAC negative) lines, but none of them were as useful as expected]. Nevertheless, we have established a Cre line #4688, carrying the DNA fragment encoding calcitonin receptor-like receptor in BAC1, in which Cre recombination occurs predominantly in the dentate mossy cells and, in part, hippocampal CA3c pyramidal cells. The dentate mossy cell is a glutamatergic excitatory cell, which receives mossy fibers from dentate granule cells and projects back to granule cells, forming disynaptic recurrent pathway in the dentate gyrus. Mossy cell loss as well as mossy fiber sprouting has been recognized after temporal lobe epilepsy. Therefore, Cre line #4688 could be useful for the study of dentate gyrus function and temporal lobe epilepsy. In order to achieve the cell-type restricted Cre expression more efficiently, we have investigated several BAC manipulation methods since October 2004, and a new approach using RED/ET system has been employed to introduce Cre cDNA just prior to the initial methionine codon in the BAC clone, to obtain a single DNA fragment that includes the promoter region and Cre cDNA. Moreover, to achieve homologous recombination in any desired BAC clones, we introduced a SacB negative selection marker (a gift from Dr. Heintz lab at Rockefeller University) to exclude the shuttle vector backbone. This BAC manipulation method for homologous recombination now enables us to efficiently introduce Cre recombinase cDNA to any desired sites of the BAC clones. We have chosen 5 additional BAC clones, each of which contain the gene product whose expression is well confined to particular brain areas as mentioned above. Currently, BAC 14-Cre for prefrontal cortex, BAC16-Cre for VTA, and BAC 20-Cre for NAcc have been injected into eggs to generate Cre transgenic mice in the Transgenic Core Facility. Cre cDNA insertion by homologous recombination into other BAC clones is also underway. Since all the lines carry BAC-derived enhancer/promoter elements and Cre cDNA, it is probable for us to obtain cell-type or region-restricted Cre transgenic mouse lines dependent upon the target gene promoter in the BAC fragment. Once Cre-lines are established from these founder mice, we crossed them with a Rosa26 reporter line, in which the expression of Cre recombinase is functionally visualized by X-gal staining. 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 (NRs). The behavioral and physiological analyses of these future NR knockout mice may hopefully lead to our understanding of the most serious neuropsychiatric disorders, such as bipolar disorder and schizophrenia. We have also initiated two additional floxed mouse projects to explore the in vivo function of particular brain areas. One is to generate transgenic mice, in which cell ablation can be induced in the cells where Cre recombinase is highly expressed, using a variant of human heparin binding-epidermal growth factor (HB-EGF) precursor as a diphtheria toxin receptor (DTR). Since diphtheria toxin administered by intra-peritoneal injection does not bind to murine HB-EGF precursor, cell ablation by DT binding will depend on the expression of human HB-EGF following Cre-loxP excision in the floxed HB-EGF alleles of mice. In collaboration with Dr. Kenji Kohno in Nara, Japan, Kazu Nakazawa and Yoko Yabe have engaged in making the construct for floxed-human HB-EGF mice. Also, we have introduced a floxed mice carrying simian HB-EGF targeted to Rosa26, the house keeping gene locus, from Drs. Ari Waisman and Thorsten Buch in Germany. We hope to cross these floxed-DTR lines with future Cre lines. The second project to generate new floxed mice targets the Kir3.2 locus. In some brain areas, such as VTA, Kir3.2-type inwardly rectified potassium channels are predominantly expressed among many other Kir channels. Based on the report showing slight membrane depolarization of VTA in the Kir3.2 KO mice, we plan to generate floxed-Kir3.2 mice, and to cross with future VTA-Cre line to depolarize VTA, thereby altering dopamine secretion into NAcc. Juan Belforte and Noelia Vargas Pinto are working on making this knock-in construct of floxed-Kir3.2. This project, in conjunction with generation of VTA-Cre line, would provide an excellent tool for the study of dopamine function in the NAcc during behavior and in vivo physiology. Finally, it is possible to use genetic protein synthesis knock-down mice with future Cre lines, in order to understand the role of protein synthesis in the particular brain areas, as described in a separate Project (# MH002846-02) from my laboratory. These additional lines of floxed mice will enable us to investigate functional roles of particular brain areas in learning and memory at a different level.

大脑的巨大复杂性源自数百种神经元细胞类型以及它们之间广泛的突触连接。最近，基因工程的发展彻底改变了大脑分区局部功能的研究，基因工程可以理想地在体内某个大脑分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人大脑有丝分裂后神经元细胞的条件转基因操作具有不可估量的价值。自2003年3月起，我们启动了创建多种脑分区或细胞类型限制性条件转基因小鼠的项目。我们今年的目标是使用携带启动子的 BAC（细菌人工染色体）克隆来创建各种亚区域限制性 Cre 重组酶转基因系，以指导特定脑区域的表达。从文献和我们的原位杂交组织化学中，我们鉴定了一些携带基因表达启动子的 BAC 克隆，这些启动子主要在海马 CA1（即 BAC7）、杏仁核（BAC1）、内嗅皮层（BAC5）、前额叶皮层（BAC4）、前脑中间神经元 (BAC6)、腹侧被盖区 (BAC8) 和伏隔核 (BAC3 和BAC10），分别。到2004年9月，在转基因核心设施（James Pickel博士）的帮助下，我们完成了将HSP70最小启动子的DNA片段-Cre cDNA和每个BAC克隆的DNA片段共注射到小鼠受精卵中，以产生双-整合转基因系。从那时起，我们花了很多时间通过与 Rosa26 报告基因 LacZ 系杂交来进行基因分型和分析 Cre 重组模式。然而，跨 BAC 克隆产生的 Cre/BAC 双转基因系比预期要少。在我们从核心设施获得的 106 个 Cre 阳性转基因品系中，只有 22 个品系也是 BAC 阳性。双转基因小鼠产量低的原因可能是由于难以制备相同拷贝数、不同长度的DNA片段。虽然我们仍在分析这 22 个品系中的 4 个，但我们没有获得任何模拟 Cre 重组酶表达到每个 BAC 基因内源表达的理想品系。 [我们还分析了 50 多个 Cre 阳性（BAC 阴性）细胞系，但没有一个像预期的那样有用]。尽管如此，我们还是建立了 Cre 系#4688，携带编码 BAC1 中降钙素受体样受体的 DNA 片段，其中 Cre 重组主要发生在齿状苔藓细胞中，部分发生在海马 CA3c 锥体细胞中。齿状苔藓细胞是一种谷氨酸能兴奋细胞，它从齿状颗粒细胞接收苔藓纤维并投射回颗粒细胞，在齿状回中形成双突触循环通路。颞叶癫痫后已发现苔藓细胞丢失和苔藓纤维发芽。因此，Cre line #4688 可用于齿状回功能和颞叶癫痫的研究。 为了更有效地实现细胞类型限制的Cre表达，我们自2004年10月起研究了几种BAC操作方法，并采用了一种使用RED/ET系统的新方法，将Cre cDNA引入到Cre cDNA的起始蛋氨酸密码子之前。 BAC 克隆，以获得包含启动子区域和 Cre cDNA 的单个 DNA 片段。此外，为了在任何所需的 BAC 克隆中实现同源重组，我们引入了 SacB 阴性选择标记（洛克菲勒大学 Heintz 博士实验室的礼物）以排除穿梭载体主链。这种用于同源重组的 BAC 操作方法现在使我们能够有效地将 Cre 重组酶 cDNA 引入 BAC 克隆的任何所需位点。我们选择了另外 5 个 BAC 克隆，每个克隆都含有基因产物，其表达很好地限制在如上所述的特定大脑区域。目前，用于前额皮质的 BAC 14-Cre、用于 VTA 的 BAC16-Cre 和用于 NAcc 的 BAC 20-Cre 已被注射到鸡蛋中，在转基因核心设施中生成 Cre 转基因小鼠。通过同源重组将 Cre cDNA 插入其他 BAC 克隆中的工作也在进行中。由于所有品系都携带BAC衍生的增强子/启动子元件和Cre cDNA，因此我们有可能获得依赖于BAC片段中的靶基因启动子的细胞类型或区域限制的Cre转基因小鼠品系。一旦从这些创始小鼠中建立了 Cre 系，我们就将它们与 Rosa26 报告系杂交，其中 Cre 重组酶的表达通过 X-gal 染色进行功能可视化。 一旦这些细胞系建立起来，我们将进一步缩小该项目的范围，将 NMDA 受体敲除针对特定的细胞类型，并研究区域限制性敲除 NMDA 受体 (NR) 的行为和生理后果。对这些未来 NR 基因敲除小鼠的行为和生理分析有望帮助我们了解最严重的神经精神疾病，例如双相情感障碍和精神分裂症。我们还启动了另外两个 floxed 小鼠项目，以探索特定大脑区域的体内功能。一种是产生转基因小鼠，其中使用人肝素结合表皮生长因子（HB-EGF）前体的变体作为白喉毒素受体（DTR），可以在Cre重组酶高表达的细胞中诱导细胞消融。由于通过腹膜内注射施用的白喉毒素不与小鼠HB-EGF前体结合，因此DT结合的细胞消融将取决于小鼠floxed HB-EGF等位基因中Cre-loxP切除后人HB-EGF的表达。 Kazu Nakazawa 和 Yoko Yabe 与日本奈良的 Kenji Kohno 博士合作，致力于制造 floxed 人类 HB-EGF 小鼠的构建体。此外，我们还引入了一只携带猿类 HB-EGF 的 floxed 小鼠，该小鼠靶向 Rosa26（管家基因座），来自 Drs.阿里·韦斯曼和托尔斯滕·布赫在德国。我们希望将这些 floxed-DTR 线与未来的 Cre 线交叉。 生成新 floxed 小鼠的第二个项目针对 Kir3.2 基因座。在某些大脑区域，例如 VTA，Kir3.2 型内向整流钾通道在许多其他 Kir 通道中主要表达。基于显示 Kir3.2 KO 小鼠中 VTA 膜轻微去极化的报告，我们计划生成 floxed-Kir3.2 小鼠，并与未来的 VTA-Cre 系杂交以去极化 VTA，从而改变多巴胺分泌到 NAcc 中。 Juan Belforte 和 Noelia Vargas Pinto 正在致力于制造这种 floxed-Kir3.2 的敲入结构。该项目与 VTA-Cre 系的产生相结合，将为研究 NAcc 在行为和体内生理学过程中的多巴胺功能提供一个极好的工具。 最后，可以使用具有未来 Cre 系的基因蛋白质合成敲除小鼠，以了解蛋白质合成在特定大脑区域中的作用，如我实验室的一个单独项目 (# MH002846-02) 中所述。这些额外的 floxed 小鼠品系将使我们能够在不同水平上研究特定大脑区域在学习和记忆中的功能作用。