Transgenic resources for neuroscience research

摘要 NIMH转基因核心设施有几个主要功能：1)为NIH的神经科学研究生产转基因产品；2)开发新的转基因技术和模型系统；3)利用神经科学遗传研究中的相关技术支持研究；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23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14个转基因大鼠项目，每个项目产生多个品系。 C)14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)发展 在过去的一年里，已经制定了允许更多生产方法的项目。两个值得注意的项目是 A)在与NIDA的合作中，CORE开发了一组大鼠，这些大鼠在针对神经元亚群的转录启动子的控制下产生重组酶或光遗传探针。已经生产了14条这样的生产线，其中8条正在由最终用户确定，并将可供分销。 B)开发生产转基因绒猴的方法。在过去的一年里，核心设施在开发生产转基因绒猴的技术方面取得了快速进展。目前已开发出生产过多胚胎的方法，优化手术程序以最有效地采集这些胚胎，培养这些胚胎，开发转基因慢病毒，感染胚胎，利用超声可视化将这些胚胎转移到受体雌性体内，并开发原代绒猴细胞的组织培养方法。这一广泛的努力已经为NIH研究人员在校内计划的基础研究中使用这些转基因动物的几个项目奠定了基础。 3)技术支持 A)通过冷冻保存生殖细胞或胚胎，已获得211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87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。 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。 。 粘脂症IV：这种疾病的小鼠模型是与斯拉根豪普特实验室长期合作的结果，并继续取得成果，包括对可能与这种疾病相关的神经病变的描述。核心设施继续分发这些动物。在转基因动物中对Mcoln1基因进行更具体的改变正在计划中。 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。 家族性自主神经障碍：与斯拉根豪普特实验室的另一项合作导致了这种疾病的模型。携带人类正常或疾病基因的品系正在核心中被创造出来。这些品系被交叉成一条空品系，用人类疾病的等价物替换内源性IKBKAP基因。

SUMMARY The NIMH transgenic core facility has several major functions: 1) to produce transgenics for neuroscience research at NIH 2) develop new transgenic techniques and model systems 3) support research with associated techniques in genetic research in neuroscience and 4) engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Meterics of production over the past year included: a) 23 transgenic mouse projects produced by oocyte injection, with multiple lines produced for each project. b) 14 transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. c) 14 mouse projects have first altering the genes of ES cell and then using those to produce mice. 2) Development Over the last year projects to allow more productive methods have been developed. Two notable projects are a) in collaboration with NIDA the core has development of a panel of rats that produce recombinases or optogenetic probes under the control of transcriptional promoters that are specific for subpopulations of neurons. Fourteen of those lines have been produced, eight of which are being characterized by end users and will available for distribution. b) developing methods to produce transgenic marmosets. Over the past year rapid progress in developing techniques to produce transgenic marmosets have been made in the core facility. The methods to produce an excess of embryos, to optimize surgical procedures to harvest those embryos most efficiently, to culture those embryos, develop transgenic lentivirus, infect embryos, transfer those embryos into recipient females using ultrasound visualization and develop tissue culture methods for primary marmoset cells have been developed. This extensive effort has set the stage for several projects for NIH researchers to use these transgenic animals in basic research in the intramural program. 3) Technical Support a) 211 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 87 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. 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. . Mucolipidosis IV: The mouse model of this disease resulted from a long-standing collaboration with the Slaugenhaupt laboratory and has continued to yield results, including a description of the neuropathy that may be associated with this disease. The core facility continues to distribute these animals. More specific alterations of the Mcoln1 gene in transgenic animals are being planned. 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. Familial dysautonomia: Another collaboration with the Slaugenhaupt lab resulted in a model for this disease. Lines carrying either a human normal or disease gene are being created in the core. These lines are crossed into a null line to replace the endogenous IKBKAP gene with its human disease equivalent.