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)通过卵母细胞注射获得48个转基因小鼠系。 B)用卵母细胞注射法建立了14个大鼠品系。 C)13个小鼠项目首先改变了胚胎干细胞的基因，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)在针对神经元亚群的转录启动子的控制下，发育出一组产生Cre重组酶的大鼠。 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，已经获得了98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68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：一种特定的基因(卡巴胆碱-O-甲基转移酶)在压力易感性中的作用在转基因小鼠中被证明是降低了该基因的水平。 神经发生：已经产生了转基因小鼠和最近的转基因大鼠，以研究成年神经发生的作用。从妊娠中期到老年，大脑中会产生新的神经元。在成人中出现的新细胞的作用特别有趣，这表明它在学习和记忆方面具有功能，并可能治疗神经退行性疾病。 精神分裂症：具有类似精神分裂症的行为特征的小鼠是通过核心设施的效应鼠与反应鼠交配而产生的。后者进行了有条件的NMDA受体的消融。这些交配的后代被设计成在皮质边缘中间神经元的子集中缺乏NMDA受体。它们在正常交配、筑巢和类似焦虑的行为上表现出行为缺陷。在这些动物身上也发现了记忆功能障碍。 压力和神经发生：最近一篇使用转基因核心培育的小鼠的论文表明，神经发生对于维持对压力的正常反应至关重要。这一发现意义重大，因为它确定了成年动物神经发生的这一角色。继其他显示压力会减少神经发生的工作之后，这一新的发现暗示了压力可能会增加的循环。 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。 药物成瘾：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老鼠来研究对成瘾药物做出反应的神经活动模式。 。 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。 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。 神经胶质活性报告：核心产生了一个转基因小鼠品系，表明神经胶质细胞中钙的浓度。通过其荧光性质的变化，已经证明了这些细胞中的钙浓度和相关活性。 报告者和效应者小鼠和大鼠：

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) 48 transgenic mouse lines produced by oocyte injection. b) 14 rat lines have been produced by oocyte injection. c) 13 mouse projects have first altering the genes of embryonic stem 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) development of a panel of rats that produce CRE recombinase under the control of transcriptional promoters that are specific for subpopulations of neurons. 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) 98 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 68 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: The role of a specific gene (catachol-O-methyltransferase) in the susceptibility to stress was demonstrated in mice that were engineered to have reduced levels of this gene. Neurogenesis: Transgenic mice and, more recently, transgenic rats have been generated to study the role of neurogenesis in adults. From mid-gestation and continuing into old age, new neurons are produced in the brain. The role of new cells appearing in adults is especially interesting, and suggests a function in learning and memory and potential treatments for neurodegenerative disease. Schizophrenia: Mice with behavioral characteristics that resemble schizophrenia were produced by mating effecter mice from the core facility with responder mice. The latter carried a conditional ablation of the NMDA receptor. The offspring of these matings were engineered to lack NMDA receptors in a subset of corticolimbic interneurons. They displayed behavioral deficits in normal mating, nest-building and anxiety-like behavior. Memory dysfunction was also revealed in these animals. Stress and neurogenesis: A recent paper using mice developed in the transgenic core has shown that neurogenesis is critical for maintaining a normal response to stress. This finding is significant because it conlusively identifies this role for neurogenesis in adult animals. Following other work that shows that stress reduces neurogenesis, this new finding implies a cycle in which stress can increase. 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. Drug addiction: 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. . 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. 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. Glial activity reporters: The core produced a mouse line with a transgene that indicated the concentration of calcium in glial cells. By changes in its fluorescent properties, the calcium concentration and associated activity has been demonstrated in these cell. Reporter and effector mice and rats: