Transgenic resources for neuroscience research

用于神经科学研究的转基因资源

• 批准号: 8745787
• 负责人: James Pickel
• 金额: $ 182.95万
• 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/8745787
• 关键词: Agreement; Animals; Archives; Behavior; Biochemical; Biological Models; Callithrix; Cells; Chimera organism; Collaborations; Complex; Conditioned Culture Media; Consult; Core Facility; Development; Disease; Embryo; FOS Family Genes; Freezing; Fright; Genes; Genetic; Genetic Recombination; Genetic Research; Germ Cells; Goals; Harvest; Health Status; Human; Human Resources; In Vitro; Inherited; Injection of therapeutic agent; Institutes; Ion Channel; Japan; Karyotype; Laboratories; Lead; Learning; Light; Link; Long-Evans Rats; Memory; Messenger RNA; Methods; Modeling; Morphology; Mus; National Institute of Dental and Craniofacial Research; National Institute of Drug Abuse; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; Neuraxis; Neurons; Neurosciences; Neurosciences Research; Oocytes; Oranges; Paper; Patients; Pattern; Peptides; Pharmaceutical Preparations; Population; Postdoctoral Fellow; Production; Protein Biosynthesis; Proteins; RNA; Rattus; Recording of previous events; Research Personnel; Research Support; Resources; Rewards; Risk; Rodent; Role; Stress; Structure; Synapses; System; Techniques; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; addiction; blastocyst; design; embryonic stem cell; experience; fascinate; improved; learned behavior; mouse model; neural circuit; neural patterning; neurogenesis; neuropsychiatry; offspring; pathogen; response; sperm cell; stem; success; trafficking; trait

SUMMARY 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 system and 4) engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Meterics of production over the past year included: a) 15 transgenic mouse projects produced by oocyte injection, with multiple lines produced for each project. b) 7 transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. c) 6 mouse projects have first altering the genes of ES cell and then using those to produce mice. 2) Technical Support a) 127 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 61 lines have been rederived, 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) Development a) transgenic marmosets: Over the last year the effort to make transgenic marmosets has increased with addition of personnel and the establishing of a collaborative agreement with Erika Sasaki at the Central Institute for Experimental Animals in Kawasaki, Japan. Because of this collaboration the methods to harvest ooctyes and blastocysts, which had been successful were further improved. Methods for in vitro methods have been completely changed to reflec them methods that are used in Dr Sasaki's laboratory. The media, the culture conditions and the analysis of ooctyes and embryos is now modelled on the successful methods that their laboratory demonstrated. The collaboration with NINDS continues, with a new postdoctoral fellow in that institute and a postBAC fellow in the core facility. b) rat ES lines: Rat ES (embryonic stem) lines, some of which ubiquitously express the orange fluorescent protein 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. At this point the karyotype of the lines will be checked before any are injected or fused to embryos in order to determine the potential to produce chimeras and transmit the OFP gene to offspring through the germline. c) Transgenic rat production: in collaboration with NIDA, the core produces transgenic rat lines that are designed in conjunction with acutely 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. d) 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, these methods will be improved. e) enhanced recombination methods: While the core has used TALENS in ES cells, we have had no success using this method in embryos. We are now collaboration with NIDA and NIDCR laboratories to develop new methods of enhanced recombination using the CRISPR/cas system. 4) Collaborative projects: below is a list of projects that have been initiated in 2011, or have continued from last year. Stress and neurogenesis: Mice produced in the transgenic core were described in a paper that showed the necessity of neurogenesis for the normal response to stress. Since it has been shown that stress reduces neurogenesis this newer result indicates that a cycle of increasing stress. 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 rrole 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 over express 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.

概括 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15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7个转基因大鼠项目，每个项目产生多个品系。 c) 6个小鼠项目首先改变ES细胞的基因，然后利用这些基因来产生小鼠。 2）技术支持 a) 通过冷冻保存生殖细胞或胚胎，已对 127 个转基因啮齿动物品系进行了存档。 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 61 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）开发 a) 转基因狨猴：去年，随着人员的增加以及与日本川崎中央实验动物研究所的 Erika Sasaki 建立合作协议，转基因狨猴的生产工作有所增加。由于这次合作，已经成功的卵母细胞和囊胚收获方法得到了进一步改进。体外方法已完全改变，以反映佐佐木博士实验室使用的方法。卵母细胞和胚胎的培养基、培养条件和分析现在均以他们实验室展示的成功方法为蓝本。与 NINDS 的合作仍在继续，该研究所有一名新的博士后研究员，核心设施有一名 BAC 后研究员。 b) 大鼠 ES 系：实验室创建了大鼠 ES（胚胎干）系，其中一些普遍表达橙色荧光蛋白。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。此时，在将任何品系注射或融合到胚胎之前，将检查品系的核型，以确定产生嵌合体并通过种系将 OFP 基因传递给后代的潜力。 c) 转基因大鼠生产：与 NIDA 合作，核心生产转基因大鼠品系，这些品系与急性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。 d) 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，但在与日本研究人员协商后，这些方法将得到改进。 e) 增强重组方法：虽然核心已在 ES 细胞中使用 TALENS，但我们在胚胎中使用此方法尚未成功。我们现在正在与 NIDA 和 NIDCR 实验室合作，开发使用 CRISPR/cas 系统增强重组的新方法。 4) 合作项目：以下是2011年启动或去年延续的项目清单。 压力和神经发生：一篇论文描述了转基因核心产生的小鼠，表明神经发生对于压力的正常反应是必要的。由于已经证明压力会减少神经发生，因此这一新的结果表明存在一个压力增加的循环。 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。过度表达具有这种结构的 mRNA 的小鼠已经被培育出来，以破坏这种易位机制。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，该机制可用于将特定消息专门发送到突触。