La Jolla Interdisciplinary Neuroscience Center Cores - Vital V

拉荷亚跨学科神经科学中心核心 - Vital V

• 批准号: 8132831
• 负责人: STUART A LIPTON
• 金额: $ 6.36万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 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/8132831
• 关键词: Accounting; Affinity; Anatomy; Area; Arts; Behavior; Biohazardous Substance; Biology; Brain; Brain region; Cancer Center; Cancer Center at the Burnham Institute; Cell Line; Cell physiology; Cells; Cellular biology; Cherry - dietary; Cloning; Collaborations; Collection; Communities; Complement; Complementary DNA; Complex; Consult; Consultations; Contracts; Core Facility; Dependovirus; Doctor of Philosophy; Engineering; Epitopes; Ferrets; Fostering; Gene Delivery; Gene Expression; Generations; Genes; Genetic; Genetic Materials; Genetic Techniques; Genome; Goals; HSV vector; Head; Housing; Human; Human Genome; Image; Individual; Injection of therapeutic agent; Institutes; Laboratories; Laboratory Organism; Length; Lentivirus Vector; Libraries; Link; Liquid substance; Location; Maintenance; Mediation; Medical Research; Methods; Mission; Molecular; Molecular Genetics; Monkeys; Mus; Neuroanatomy; Neurons; Neurophysiology - biologic function; Neurosciences; Normal Cell; Oligonucleotides; Pathway interactions; Phosphotransferases; Physiology; Plasmids; Preparation; Price; Procedures; Production; Protein Family; Protein Kinase; Proteins; Proteome; Protocols documentation; RNA Interference; Rattus; Reagent; Research Personnel; Robotics; Screening procedure; Services; Signal Transduction; Simplexvirus; Small Interfering RNA; Source; Structure; Subfamily lentivirinae; Technical Expertise; Technology; Testing; Time; Toxic effect; Transfection; Transgenic Mice; Transgenic Organisms; Vendor; Vertebral column; Viral; Viral Vector; attenuation; base; cDNA Expression; cancer cell; design; experience; gene function; genetic manipulation; genetic technology; high throughput screening; human disease; in vivo; insight; material transfer agreement; mature animal; meetings; member; neurophysiology; novel; positional cloning; promoter; research study; small hairpin RNA; small molecule libraries; structural biology; tool; vector; virology

VIRAL VECTOR AND siRNA CORE FACILITY 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Viral Vector/siRNA Core will be a new facility. Currently, there is no unified Core facility to link neuroscientists on the La Jolla Torrey Pines Mesa for these functions. Therefore, this facility will constitute a new Core to foster the use of viral vectors for payload delivery into neural cells, including RNAi, among neuroscientists and will produce multiple collaborations in an interdisciplinary manner in the San Diego area on the La Jolla Torrey Pines Mesa. The primary core will be located at The Salk Institute because of the presence of a prominent virologist/geneticist there, Dr. Inder Verma, who will Direct the Core with the assistance of Edward Callaway, who will act as Co-Director. A satellite facility will be located at the Burnham Institute for Medical Research (BIMR) headed by Mark Mercola, who will serve as a Co-Director for the core. This satellite is particularly important for seamless interface with the other cores located there that will use the viral vectors, e.g., the Chemical Library Screening/High Throughput Cell Analysis Cores and the Imaging Core. The mission of the Viral Vector and siRNA Core Facility (WS) is to provide a robust means of gene manipulation to Neuroscience investigators. Over the last decade, methods for the production of a variety of viral vectors for stable and non-toxic delivery of genetic material to postmitotic neurons have become relatively routine. However, the laboratories that would most benefit from the use of these vectors often have expertise in neuroanatomy, neurophysiology, or behavior, but not virology. This disparity between expertise and need greatly limits the use of these valuable vectors within the San Diego Neuroscience community. Furthermore, even those laboratories that have the technical capability to produce one or another type of vector would benefit from a core facility which maintains proven protocols, technical expertise and experience, and all the necessary plasmids required to implement the full range of possibilities afforded by modern virology. The San Diego neuroscience community presently has the expertise to establish and maintain a state-of-the art facility, but piecemeal collaboration amongst a limited set of laboratories does not fully realize the potential of this community. Establishing a facility to make a broad range of vectors including adeno-associated virus (AAV), lentivirus, and herpes simplex virus (HSV) amplicon vectors will have fundamental impact on the culture of Neuroscience in San Diego. This core will make possible experiments that would be otherwise impossible and will foster collaborations amongst molecular neuroscientists who routinely use genetic methods, and other neuroscientists who focus on behavior, anatomy or physiology. Viral vectors are now fundamental tools at the cutting edge of experimental neuroscience. This is because they harness the proven power of molecular and genetic techniques by complementing and extending the capabilities afforded by the production of transgenic mice. Amongst the most prominent advantages of viral vectors are: 1) the short time required to test a genetic manipulation (e.g. gene knockdown with siRNA) compared to the generation of a transgenic mouse line; 2) the ability to target gene expression to particular brain regions where specific promoters are not available for targeting; 3) the ability to initiate manipulation in adult animals; 4) the ability to use genetic technologies in species where transgenic methods are impractical (e.g., monkeys). Nevertheless, each of the available viral vector technologies have limitations, such that no single vector will meet the needs of every possible experimental goal. We have selected three types of viral vectors, AAV, lentivirus, and HSV amplicons, because these are all proven to be highly effective and they are a complementary set. Limitations of one vector can often be obviated by the use of another. These vectors all have in common, however, that they can efficiently transduce nearly every neuron in the vicinity of a brain injection and they can yield stable gene expression for months or years without toxicity (Naldini et al., 1996; Blomer et al., 1997; Xiao et al., 1997; Rabinowitz and Samulski, 1998; Sandier et al., 2002; Davidson and Breakefield, 2003; Kootstra and Verma, 2003). In addition to supporting the construction and use of viral vectors, the core will also provide reagents and expertise in gene knockdown by si/shRNA. Gene attenuation has been central to experimental biology for the past century and has increased exponentially with the advent of reverse genetics or the use of targeted attenuation of gene activity to reveal function. It is anticipated that gene attenuation studies performed at a large, genome or proteome-wide scale, will be increasingly important to assign gene function now that the sequences of many experimental organisms are complete. Moreover, gene function will point to targets that will be interrogated further using other technologies embodied in the other Neuroscience Cores, including Chemical Library Screening and Structural Biology; thus, this core is an important component of an integrated approach to Neurosciences. Currently, there is no si/shRNA and viral vector facility or core service available to Neuroscientists. Individual laboratories contract with commercial entities to produce siRNA oligos or construct si or shRNA vectors internally and viral vector technology is represented at a very sophisticated level in a few laboratories. The Cancer Center at BIMR is establishing a facility that will distribute a commercial library of siRNA oligonucleotides against human genome targets, but this will be available only to Cancer Center members, and efficacy of transfection of siRNA oligonucleotides into many cell lines, especially primary cell lines, is limited. The core facility will offer 1) AAV, lenti and HSV vector support, 2) access to a commercial siRNA oligonucleotide library for screening, 3) access to a shRNA lentiviral library to the human kinome, 4) . shRNA lentiviral libraries to other targets will be offered as libraries become available; in addition, the core will offer: 5) tagged human cDNA clones representing the entire kinome in a 3rd generation lentiviral vector and 6) a negotiated preferred price structure for purchasing siRNA oligonucleotides from a commercial vendor. Kinases have been chosen for the first shRNA and expression lentiviral libraries to be offered by the facility because they constitute one of the largest and most important of protein families, accounting for ~2% of genes in human and other eukaryotic genomes (Manning et al., 2002a; Manning et al., 2002b). By phosphorylating substrate proteins, kinases modify the activity, location, and affinities of up to 30% of all cellular proteins, and direct most cellular processes, particularly in signal transduction and coordination of complex pathways. In fact, it is difficult to think of any pathway that is not modulated by kinases, making them, as a group, a lynchpin of cell biology, and promising that a set of high quality knockdown reagents would provide insight into the control of almost any cellular process. The human genome contains 518 kinase genes, of which over 150 have already been implicated in human disease (Manning, 2005). The WS facility will be housed at the Salk Institute with a satellite facility at the BIMR. The purpose of the BIMR satellite is to have access to the robotic liquid handling capacity of the Chemical Library Screening (CLS) to amplify and array the libraries. The Salk facility will be under the direction of Inder Verma, PhD and Ed Callaway, PhD as co-director. Dr. Verma's laboratory has dedicated the last 30 years to understanding the molecular underpinnings that convert a normal cell to a cancer cell. He has developed expertise in large-scale manipulation of lentiviral vectors for the expression of cDNAs and si/shRNA. Dr. Callaway is an expert in cortical neural function and organization and his lab has expertise in the production and use of AAV, lentiviral and HSV amplicon vectors for gene delivery. The lab also has extensive experience in using these vectors in vivo in monkeys, ferrets, rats and mice.

病毒载体和 siRNA 核心设施 一、主要目标和新方向 病毒载体/siRNA 核心将是一个新设施。目前还没有统一的核心设施来链接 拉霍亚托利松台地的神经科学家研究了这些功能。因此，该设施将 构成一个新的核心，以促进使用病毒载体将有效负载传递到神经细胞中，包括 RNAi 与神经科学家之间将以跨学科的方式开展多项合作 拉霍亚托利松梅萨 (La Jolla Torrey Pines Mesa) 的圣地亚哥地区。主要核心区将位于索尔克 研究所是因为那里有一位著名的病毒学家/遗传学家 Inder Verma 博士，他将 在爱德华·卡拉威 (Edward Callaway) 的协助下指导核心工作，他将担任联合主任。卫星设施 将设在伯纳姆医学研究所 (BIMR)，由 Mark Mercola 领导，他将 担任核心联合主任。这颗卫星对于与 位于那里的其他核心将使用病毒载体，例如化学库筛选/高 吞吐量细胞分析核心和成像核心。 病毒载体和 siRNA 核心设施 (WS) 的使命是提供一种稳健的基因检测手段 对神经科学研究人员的操纵。在过去的十年中，生产方法 用于将遗传物质稳定且无毒地递送至有丝分裂后神经元的多种病毒载体 变得相对常规。然而，最能从这些技术的使用中受益的实验室 载体通常具有神经解剖学、神经生理学或行为学方面的专业知识，但不具备病毒学方面的专业知识。这 专业知识和需求之间的差距极大地限制了这些有价值的载体在 San 迭戈神经科学社区。此外，即使是那些有技术能力的实验室 生产一种或另一种类型的载体将受益于保持经过验证的核心设施 协议、技术专长和经验，以及实施该计划所需的所有必要的质粒 现代病毒学提供了全方位的可能性。圣地亚哥神经科学界目前 拥有建立和维护最先进设施的专业知识，但之间的零碎合作 有限的实验室并不能充分发挥这个社区的潜力。建立设施以 制作多种载体，包括腺相关病毒 (AAV)、慢病毒和单纯疱疹病毒 病毒（HSV）扩增子载体将对圣地亚哥的神经科学文化产生根本性影响。 这个核心将使原本不可能进行的实验成为可能，并将促进 经常使用遗传方法和其他方法的分子神经科学家之间的合作 专注于行为、解剖学或生理学的神经科学家。 病毒载体现在是实验神经科学前沿的基本工具。这是 因为它们通过补充和利用分子和遗传技术的经过验证的力量 扩展转基因小鼠生产所提供的能力。其中最突出的 病毒载体的优点是：1）测试基因操作（例如基因）所需的时间短 siRNA敲除）与转基因小鼠系的产生进行比较； 2）目标能力 基因表达到特定的大脑区域，其中特定的启动子无法用于靶向； 3） 对成年动物进行操纵的能力； 4）在物种中使用遗传技术的能力 转基因方法不切实际的地方（例如猴子）。尽管如此，每种可用的病毒 载体技术有其局限性，因此没有任何一种载体能够满足所有可能的需求 实验目标。我们选择了三种类型的病毒载体，AAV、慢病毒和HSV扩增子， 因为这些都被证明是非常有效的，而且它们是互补的。一的局限性 通常可以通过使用另一个向量来避免。然而，这些向量都有一个共同点： 它们可以有效地转导大脑注射附近的几乎每个神经元，并且可以产生 基因稳定表达数月或数年而无毒性（Naldini 等人，1996；Blomer 等人，1997；Xiao 等，1997；拉比诺维茨和萨穆尔斯基，1998；桑迪尔等人，2002；戴维森和布雷克菲尔德，2003； 库斯特拉和维尔玛，2003）。 核心除了支持病毒载体的构建和使用外，还将提供试剂和 si/shRNA 基因敲除方面的专业知识。基因衰减一直是实验生物学的核心 在过去的一个世纪中，随着反向遗传学的出现或使用 有针对性地减弱基因活性以揭示功能。预计基因衰减研究 在大型基因组或蛋白质组范围内进行，分配基因将变得越来越重要 现在许多实验生物体的序列已经完成。此外，基因 功能将指向将使用包含在其中的其他技术进一步询问的目标 其他神经科学核心，包括化学库筛选和结构生物学；因此，这个核心 是神经科学综合方法的重要组成部分。 目前，神经科学家没有可用的 si/shRNA 和病毒载体设施或核心服务。 个别实验室与商业实体签订合同生产 siRNA 寡核苷酸或构建 si 或 shRNA 载体内部和病毒载体技术在一些领域处于非常复杂的水平 实验室。 BIMR 癌症中心正在建立一个设施，用于分发商业图书馆 针对人类基因组目标的 siRNA 寡核苷酸，但这仅适用于癌症 中心成员，以及将 siRNA 寡核苷酸转染到许多细胞系中的功效，特别是 原代细胞系是有限的。 核心设施将提供 1) AAV、慢病毒和 HSV 载体支持，2) 获得商业 siRNA 用于筛选的寡核苷酸文库，3) 获得人类激酶组的shRNA慢病毒文库，4)。 当文库可用时，将提供针对其他靶标的 shRNA 慢病毒文库；此外， 核心将提供：5) 代表第三代慢病毒中整个激酶组的标记人类 cDNA 克隆 载体和 6) 从以下公司购买 siRNA 寡核苷酸的协商优先价格结构 商业供应商。 激酶已被选为第一个 shRNA 和表达慢病毒文库，由 设施，因为它们构成了最大和最重要的蛋白质家族之一，占 人类和其他真核生物基因组中约 2% 的基因（Manning 等人，2002a；Manning 等人，2002b）。 通过磷酸化底物蛋白，激酶可改变高达 30% 的底物蛋白的活性、位置和亲和力。 所有细胞蛋白质，并指导大多数细胞过程，特别是在信号转导和 复杂路径的协调。事实上，很难想象任何途径不受以下因素的调节： 激酶，使它们作为一个整体成为细胞生物学的关键，并承诺提供一系列高质量的 敲除试剂将提供对几乎所有细胞过程的控制的深入了解。人类 基因组包含 518 个激酶基因，其中 150 多个已与人类疾病有关 （曼宁，2005）。 WS 设施将设在索尔克研究所，并在 BIMR 设有卫星设施。目的 BIMR 卫星将能够使用化学图书馆的机器人液体处理能力 筛选（CLS）以扩增和排列文库。索尔克工厂将由 Inder 领导 Verma 博士和 Ed Callaway 博士担任联合主任。 Verma 博士的实验室奉献了过去 30 多年的时间来了解将正常细胞转化为癌细胞的分子基础。他有 开发了大规模操作慢病毒载体以表达 cDNA 的专业知识， si/shRNA。 Callaway 博士是皮质神经功能和组织方面的专家，他的实验室拥有专业知识 用于基因传递的 AAV、慢病毒和 HSV 扩增子载体的生产和使用。实验室还 在猴子、雪貂、大鼠和小鼠体内使用这些载体方面拥有丰富的经验。