Transgenic resources for neuroscience research

摘要 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产品，2)支持神经科学遗传研究中的相关技术研究，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 过去一年的产量包括： 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，每个项目产生多个系。 C)6个小鼠项目首先改变了ES细胞的基因，然后用这些基因生产小鼠。 2)技术支持 A)通过冷冻保存生殖细胞或胚胎，已获得127个转基因啮齿动物品系。 B)通过将携带病原体的动物的品系转移到具有明确健康状态的动物身上，重新获得了61个品系。 C)对于没有生产转基因动物经验的NIH神经科学实验室来说，转基因项目的设计和援助仍然具有重要意义。 3)发展 A)转基因绒猴：去年，由于增加了工作人员，并与日本川崎中央实验动物研究所的佐佐木Erika Sasaki达成了合作协议，制造转基因绒猴的努力有所增加。由于这一合作，已经成功的卵子和囊胚的收获方法得到了进一步的改进。体外方法已经完全改变，以适应佐佐木博士实验室使用的方法。现在，卵子和胚胎的培养液、培养条件和分析都是以他们实验室展示的成功方法为模型的。与NINDS的合作仍在继续，该研究所有一名新的博士后研究员，核心设施有一名博士后研究员。 B)大鼠ES系：实验室已经创造出大鼠ES(胚胎干细胞)系，其中一些普遍表达橙色荧光蛋白。这些来自Long Evans大鼠的细胞系已经培养了几代，仍然表达OFP，并保持了代表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。在这一点上，在将任何一个注入或融合到胚胎之前，将检查这些品系的核型，以确定产生嵌合体的可能性，并通过生殖系将OFP基因传递给后代。 C)转基因大鼠的生产：与NIDA合作，CORE生产转基因大鼠品系，这些品系是与尖锐地输送的转基因一起设计的，以在离散的中枢神经系统神经元群体中表达基因。这些品系是在核心设施中生产的，然后在NIDA实验室中筛选有用的表达模式。 D)支助技术：正在开发若干技术，以提高核心支助职能的能力。冷冻小鼠精子和使用新方法改善试管受精是一项主要努力。冷冻大鼠精子并在可接受的水平上完成试管受精在所有实验室都是一项具有挑战性的任务，但在咨询了日本的调查人员后，这些方法将得到改进。 E)增强重组方法：虽然核心已经在ES细胞中使用了TALENS，但我们在胚胎中使用这种方法还没有成功。我们现在正在与NIDA和NIDCR实验室合作，利用CRISPR/CAS系统开发增强重组的新方法。 4)合作项目：以下是2011年启动的或从去年开始继续的项目清单。 压力和神经发生：在一篇论文中描述了在转基因核心中产生的小鼠，这表明了正常的应激反应需要神经发生。由于已经表明压力会减少神经发生，这一新的结果表明，压力的增加是一个循环。 学习和记忆：研究了特定和严格控制的蛋白质合成对学习和记忆的影响。此外，转基因小鼠模型已经被用来显示特定的多肽表达细胞在影响恐惧、行为和学习之间的联系方面所起的作用。 操纵电路：已经为两个独立的实验室制造了小鼠，这两个实验室有特定的神经元，这些神经元可以通过光激活的离子通道暂时不活动。这些实验室正在研究活跃在学习和成瘾中的不同神经回路。 成瘾和奖赏行为：在核心设施中产生了转基因大鼠系，这些转基因大鼠在传入输入激活FOS基因的情况下表达GFP。布鲁斯·霍普斯在NIDA的实验室正在使用这些大鼠来研究神经活动对成瘾药物的反应模式，以及最近压力在减少奖赏行为重建中的作用。 神经元中的信使核糖核酸运输：RNA干环结构对于将信息转移到神经元的特定细胞隔间是必要的。为了破坏这种易位机制，已经产生了过度表达这种结构的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在正常功能中的作用。此外，这种机制可能有助于将特定的消息专门定向到突触。

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the genes of ES cell and then using those to produce mice. 2) Technical Support a) 127 transgenic rodent lines have been archived by cryopreserving germ cells or embryos.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.