Transgenic resources for neuroscience research

用于神经科学研究的转基因资源

• 批准号: 8940169
• 负责人: James Pickel
• 金额: $ 198.48万
• 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/8940169
• 关键词: 22q; Affect; Alleles; Animal Experimentation; Animals; Archives; Autistic Disorder; Behavior; Binding; Biochemical; Biological Models; Brain; Callithrix; Cells; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Complex; Consult; Core Facility; Development; Disease; Drug abuse; Embryo; FOS Family Genes; Fertilization; Freezing; Fright; Functional disorder; Gene Targeting; Gene-Modified; Genes; Genetic; Genetic Engineering; Genetic Research; Germ Cells; Goals; Guide RNA; Harvest; Health Status; Human; In Vitro; Individual; Injection of therapeutic agent; Institutes; Ion Channel; Japan; Knowledge; Laboratories; Lead; Learning; Light; Link; Long-Evans Rats; Mammals; Massachusetts; Mediating; Memory; Mental disorders; Messenger RNA; Methods; Metric; Morphology; Mus; National Institute of Dental and Craniofacial Research; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; Nature; Nervous System Physiology; Nervous system structure; Neuraxis; Neurons; Neurosciences; Neurosciences Research; Oocytes; Oranges; Ovary; Pathology; Patients; Pattern; Peptides; Pharmaceutical Preparations; Plasmids; Play; Population; Primates; Process; Production; Protein Biosynthesis; Proteins; RNA; Rattus; Recording of previous events; Research; Research Personnel; Research Support; Resources; Rewards; Ribonucleases; Rodent; Rodent Model; Role; Site; Spain; Staging; Stress; Structure; Symptoms; Synapses; Syndrome; System; Techniques; Technology; Tissues; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Translations; United States National Institutes of Health; Variant; Work; addiction; autism spectrum disorder; base; calcium indicator; density; design; embryonic stem cell; experience; genetic risk factor; genetic variant; improved; learned behavior; mouse model; neural circuit; neural patterning; neuropsychiatry; new technology; nonhuman primate; nuclease; offspring; overexpression; pathogen; response; risk variant; social; sperm cell; stem; synaptic function; trafficking; transmission process

The NIMH transgenic core facility has several major functions: 1) to produce transgenics for neuroscience research, 2) support research with associated techniques in genetic research in neuroscience, 3) develop new transgenic techniques and model systems and 4) engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year included: a) 12 transgenic mouse projects produced by oocyte injection, with multiple lines produced for each project. b) Ten transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. c) Seven mouse projects first altering the genes of embryonic stem (ES) cells, and then using those to produce mice. 2) Technical Support a) 96 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 25 lines have been re-derived, by transferring lines from pathogen bearing animals into those with defined health status. c) Transgenic project design and assistance have continued to be significant to NIH neuroscience labs without experience in producing transgenic animals. 3) Technical development a) Nuclease mediated genetic engineering: Over the last year the core facility has worked on several projects that use the CRISPR/Cas9 system to modify genes in cells and in animals. In collaboration with labs at the National Insitute on Drug Abuse (NIDA), we have investigated the function of different iterations of the CRISPR/Cas9 system in rats. With Nick Ryba in the National Institute of Dental and Craniofacial Research (NIDCR), we have worked on mice, and with Guoping Feng of the McGovern Institute at Massachusetts Institute of Technology (MIT) we have targeted genes in marmosets. Using this system across species has given the core the opportunity to generalize these methods. This includes optimizing concentration of the injected material, the site of injection in embryos, and the construction of targeting plasmids. b) Transgenic marmosets: The core's collaboration with Erika Sasaki at the Central Institute for Experimental Animals in Kawasaki, Japan continue this year. Methods for harvesting oocytes, in vitro maturation and fertilization are based with those used in Dr Sasaki's laboratory. Cooperation between the Core and Afonso Silva's laboratory in the National Institute of Neurological Disorders and Stroke (NINDS) also continues in an effort to produce marmosets that express the genetically encoded calcium indicator (GECI). Most recently a project using CRISPR/Cas9 technologies to targeted behaviorally relevant genes in marmoset oocytes (see below under collaboration). c) Rat ES lines: Rat ES lines, some of which ubiquitously express the orange fluorescent protein (OFP) have been created in the lab. These lines from Long Evans rats have been cultured for several passages, still express OFP and maintain a morphology that is representative of ES cells. Especially with rat ES lines this is not enough to insure that these lines will contribute to a chimeric animal. The OFP marker will allow the potential contribution of these ES cells to the different tissues of the chimeric animals, as well as germline transmission to offspring of these founder animals. d) Transgenic rat production: in collaboration with NIDA, the core produces transgenic rat lines that are designed in conjunction with transiently delivered transgenes to express genes in discrete populations of central nervous system neurons. These lines are produced in the core facility and then screened for useful expression patterns in NIDA laboratories. This year, four of these lines have been characterized to the point where they are in use in laboratories in NIMH, NINDS and NIDA. We have been collaborating to use the CRISPR/Cas9 system to target specific genes as well as to increase the efficiency of targeting with replacement sequences directed to specific genetic loci. e) Support techniques: several techniques are under development to increase the capacity of the core's support functions. Freezing mouse sperm and improving IVF by using newer methods is a major effort. Freezing rat sperm and completing IVF at an acceptable level is a challenging task in all laboratories, but having consulted with investigators in Japan and Spain, these methods will be improved. 4) Collaborative projects: Targeting an autism gene in non-human primates: Autism spectrum disorders have been linked to several genes but Phelan-McDermid Syndrome has been most closely associated with a single gene: SHANK3. Though other genes in the telomeric region of chromosome 22q my also be involved in creating or effecting the symptoms of the disease. The protein coded by SHANK3 is a component of the post synaptic density (PSD) and incorporates multiple structural motifs that bind other PSD proteins (SH3, PDZ, ankrin, Homer-binding regions). These features indicate that SHANK3 plays a role in the structure as well as the function of the synapse. In mice the inactivation of Shank3 causes deficits in social and repetitive behaviors as well as electrophysiological and circuit disruptions (Peca et al. Nature 472:437, 2011). Rodent models, including the one cited above have been useful in studying the conserved behaviors and functions of mammals, but the spectrum of autism diseases have a range that is lost in the translation of experimental results from rodents to primates. To build on their work in rodents, Guoping Feng's laboratory at the McGovern Institute at the MIT has developed a nuclease-mediated targeting CRISPR/Cas9 system that we have injected into marmoset oocytes. These oocytes have been harvested from the ovary of superovulated donors and allowed to mature in vitro to a specific developmental stage, been fertilized in vitro (IVF), injected with the targeting guide RNAs and the nuclease that specifically cuts the Shank3 locus. These embryos have been analyzed, and the SHANK3 locus is indeed disrupted. Learning and memory: The effect of specific and tightly controlled protein synthesis on learning and memory was studied. In addition, transgenic mouse models have been used to show the role of specific peptide-expressing cells to influence the link between fear and behavior and learning. Manipulating circuitry: Mice have been produced for two separate laboratories which have specific neurons that could be rendered transiently inactive by light activated ion channels. Those laboratories are investigating different neural circuits that are active in learning and addiction. Addictive and reward behavior: Lines of transgenic rats that express GFP in response to afferent input activation of the fos gene were generated in the core facility. These rats are being used by Bruce Hopes laboratory in NIDA to study patterns of neural activity in response to addictive drugs and most recently in the role of stress in reducing the re-establishment of rewarded behavior. mRNA trafficking in neurons: An RNA stem loop structure is necessary for the translocation of message to specific cell compartments of the neuron. Mice that overexpress mRNA with this structure have been produced in an effort to disrupt this translocation machinery. By expressing this transgenic mRNA in different neuronal subtypes, the role for this mechanism for normal function is being studied. In addition, this mechanism could be useful to target specific messages specifically to the synapse.

NIMH 转基因核心设施有几个主要功能：1）为神经科学研究生产转基因；2）支持神经科学研究遗传研究相关技术的研究；3）开发新的转基因技术和模型系统；4）参与促进神经科学研究遗传方法的合作项目。 1）生产 过去一年的生产指标包括： a) 通过卵母细胞注射产生12个转基因小鼠项目，每个项目产生多个品系。 b) 通过卵母细胞注射产生十个转基因大鼠项目，每个项目产生多个品系。 c) 七个小鼠项目首先改变胚胎干（ES）细胞的基因，然后利用它们来培育小鼠。 2）技术支持 a) 96个转基因啮齿动物品系已通过冷冻保存生殖细胞或胚胎进行存档。 b) 通过将带有病原体的动物的品系转移到具有明确健康状况的动物中，重新衍生了 25 个品系。 c) 转基因项目设计和援助对于没有生产转基因动物经验的 NIH 神经科学实验室仍然具有重要意义。 3）技术开发 a) 核酸酶介导的基因工程：去年，核心设施开展了多个使用 CRISPR/Cas9 系统修改细胞和动物基因的项目。我们与国家药物滥用研究所 (NIDA) 的实验室合作，研究了 CRISPR/Cas9 系统不同迭代在大鼠中的功能。我们与美国国家牙科和颅面研究所 (NIDCR) 的 Nick Ryba 一起对小鼠进行了研究，并与麻省理工学院 (MIT) 麦戈文研究所的冯国平一起研究了狨猴的基因。跨物种使用该系统使核心有机会推广这些方法。这包括优化注射材料的浓度、胚胎中的注射部位以及靶向质粒的构建。 b) 转基因狨猴：今年，核心团队继续与日本川崎中央实验动物研究所的 Erika Sasaki 进行合作。采集卵母细胞、体外成熟和受精的方法基于佐佐木博士实验室使用的方法。 Core 与国家神经疾病和中风研究所 (NINDS) 的 Afonso Silva 实验室之间的合作也在继续，致力于培育表达基因编码钙指示剂 (GECI) 的狨猴。最近的一个项目使用 CRISPR/Cas9 技术来靶向狨猴卵母细胞中的行为相关基因（见下文合作）。 c) 大鼠 ES 系：实验室创建了大鼠 ES 系，其中一些普遍表达橙色荧光蛋白 (OFP)。这些来自 Long Evans 大鼠的细胞系已培养数代，仍然表达 OFP 并保持代表 ES 细胞的形态。特别是对于大鼠 ES 系，这不足以确保这些系将有助于嵌合动物。 OFP 标记将允许这些 ES 细胞对嵌合动物的不同组织做出潜在贡献，以及生殖系传递给这些创始动物的后代。 d) 转基因大鼠生产：与 NIDA 合作，核心生产转基因大鼠品系，这些品系与瞬时传递的转基因结合设计，以在中枢神经系统神经元的离散群体中表达基因。这些细胞系在核心设施中生产，然后在 NIDA 实验室筛选有用的表达模式。今年，其中 4 条产品线已经过鉴定，可在 NIMH、NINDS 和 NIDA 的实验室中使用。我们一直在合作使用 CRISPR/Cas9 系统来靶向特定基因，并通过针对特定基因位点的替换序列来提高靶向效率。 e) 支持技术：一些技术正在开发中，以提高核心支持功能的能力。使用更新的方法冷冻小鼠精子并改善体外受精是一项重大努力。在所有实验室中，冷冻大鼠精子并以可接受的水平完成体外受精都是一项具有挑战性的任务，但在与日本和西班牙的研究人员协商后，这些方法将得到改进。 4）合作项目： 针对非人类灵长类动物的自闭症基因：自闭症谱系障碍与多个基因有关，但费兰-麦克德米德综合症与单个基因密切相关：SHANK3。尽管 22q 染色体端粒区域的其他基因也可能参与产生或影响该疾病的症状。 SHANK3 编码的蛋白质是突触后密度 (PSD) 的组成部分，并包含多个结合其他 PSD 蛋白质的结构基序（SH3、PDZ、ankrin、Homer 结合区）。这些特征表明 SHANK3 在突触的结构和功能中发挥着重要作用。在小鼠中，Shank3 失活会导致社交和重复行为缺陷以及电生理和电路中断（Peca et al. Nature 472:437, 2011）。 啮齿动物模型，包括上面引用的模型，对于研究哺乳动物的保守行为和功能很有用，但自闭症疾病的范围在将实验结果从啮齿动物转化为灵长类动物时丢失了。为了以啮齿类动物研究为基础，麻省理工学院麦戈文研究所冯国平实验室开发了一种核酸酶介导的靶向 CRISPR/Cas9 系统，我们已将其注射到狨猴卵母细胞中。这些卵母细胞是从超排卵供体的卵巢中采集的，并在体外成熟到特定的发育阶段，进行体外受精 (IVF)，注射靶向引导 RNA 和特异性切割 Shank3 基因座的核酸酶。这些胚胎经过分析，SHANK3 基因座确实被破坏。 学习和记忆：研究了特定且严格控制的蛋白质合成对学习和记忆的影响。此外，转基因小鼠模型已被用来展示特定肽表达细胞在影响恐惧与行为和学习之间的联系方面的作用。 操纵电路：已经为两个独立的实验室培育了小鼠，这些实验室具有特定的神经元，可以通过光激活的离子通道使其暂时失活。这些实验室正在研究在学习和成瘾方面活跃的不同神经回路。 成瘾和奖赏行为：在核心设施中生成了表达 GFP 的转基因大鼠品系，以响应 fos 基因的传入输入激活。 NIDA 的 Bruce Hopes 实验室正在使用这些大鼠来研究对成瘾药物的反应的神经活动模式，以及最近研究压力在减少奖励行为重建中的作用。 神经元中的 mRNA 运输：RNA 茎环结构对于将信息易位到神经元的特定细胞区室是必要的。为了破坏这种易位机制，人们已经培育出了过度表达具有这种结构的 mRNA 的小鼠。通过在不同的神经元亚型中表达这种转基因 mRNA，人们正在研究这种机制对正常功能的作用。此外，这种机制可用于将特定消息专门发送到突触。