Name: lentivirus-mediated genetic manipulation and visualization of olfactory sensory neurons in vivo
Uploaded: 2011-05-22T14:00:00
Description: Watch this Scientific Journal Video about Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo at JoVE.com

支持这项研究是由美国国立卫生研究院DC052256和DC006015和NSF 0324769 Qg和T32 - DC008072符合BS。

1。制备 此过程是生物安全2级，因此，显微注射过程中的生物危害罩以下所有的准备工作 。 <ol><li>准备和预热了37 ° C水床：装满水的塑料储物袋 - 清空加热块孵化器的密封和设置。这将使小鼠恢复注射后。 </li><li>填写一个生物危险废物容器的10％漂白水浸泡的所有从这个过程中浪费的一个合适的音量。 </li><li>准备一个小型开放的菜70％的乙醇和在生物安全柜的灭菌d...

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	<li>Chen, H., Kohno, K., Gong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+ablation+of+mature+olfactory+sensory+neurons+mediated+by+diphtheria+toxin+receptor.">Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor.</a> J Neurocytol. 34, 1-2 (2005).</li><li>Doi, K., Nibu, K., Ishida, H., Okado, H., Terashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+olfactory+epithelium+and+olfactory+bulb:+a+long-term+study.">Adenovirus-mediated gene transfer in olfactory epithelium and olfactory bulb: a long-term study.</a> Ann Otol Rhinol Laryngol. 114, 629-633 (2005).</li><li>Lois, C., Hong, E. J., Pease, S., Brown, E. J., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+and+tissue-specific+expression+of+transgenes+delivered+by+lentiviral+vectors.">Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.</a> Science. 295, 868-872 (2002).</li><li>Zhao, H., Otaki, J. M., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+olfactory+neurons+in+vivo.">Adenovirus-mediated gene transfer in olfactory neurons in vivo.</a> J Neurobiol. 30, 521-530 (1996).</li></ol>

微量注射慢病毒基因结构的永久转会到OSN的基因组DNA的结果。这种方法可以让我们的候选基因进行短期或长期的操作，通过过表达或shRNA介导击倒。此外，我们可以在现有的转基因小鼠，观察气味受体种群的合作本地化荧光标签单OSNs。显微注射是必需的慢病毒转导OSNs。我们曾试图进入鼻腔冲洗慢病毒悬浮导OSNs没有任何成功。因此，我们执行microinjections通过背鼻子表面，以提供病毒的OE OSN的细胞?...

显微注射：新生小鼠显微注射使用5μL注射器汉密尔顿（汉密尔顿＃7647-01）装有33号针头（汉密尔顿＃7762-06）。 免疫组织化学试剂：一抗：兔多克隆抗体的GFP（分子探针＃A - 6455），鸡对OMP的抗体（自定义）（陈等人，2005年），豚鼠对VGlut2多克隆抗体（Millipore公司＃AB2251）。二抗：CY2共轭驴抗兔（杰克逊Immunolab＃711-225-152），共轭Cy3标记的驴抗鸡（杰克逊Immunolab＃703-165-155）和Cy5标记的山羊抗豚鼠（杰克逊Immunolab＃106-175-008）。被安装在玻片Fluoromount G（南方生物技术＃0010-01）50ng/mL的DAPI染色染色解决方案。 共焦成像：Z - Stack的图像，使用奥林巴斯Flouview FV1000共聚焦显微镜，到使用奥林巴斯FV10 - ASW的2.01焦采集和分析软件的3D预测收集和处理的图像。

lentivirus-mediated genetic manipulation and visualization of olfactory sensory neurons in vivo

一个精确的嗅觉电路的发展依赖于嗅觉的感觉神经元（OSN）轴突在嗅球（OB），突触目标准确的预测。 OSN的轴突的生长和定位的分子机制不能很好地理解。操纵基因的表达和随后的单一的OSN的轴突和他们的终端乔木形态的可视化迄今已具有挑战性。要在规定的时限内的单细胞水平上研究基因的功能，我们开发了基于慢病毒的技术，操纵在体内OSNs的基因表达。慢病毒颗粒，通过显微注射到嗅上皮（OE）的OSNs。表达盒，然后永久地整合到转OSNs基因组。绿色荧光蛋白表达确定感染OSNs，并概述了他们的整个形态，包括轴突终端乔木。由于显微注射和记者检测之间的周转时间短，基因功能研究，可以集中在一个很窄的发展时期。使用这种方法，我们三天内检测GFP的表达，只要3个月以下注射。我们已经取得的过度表达和shRNA慢病毒显微注射介导击倒。这种方法提供了详细的形态在体内的单个细胞水平OSN胞体和轴突，从而使候选基因在嗅觉发展功能特性。

Watch this Scientific Journal Video about Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo at JoVE.com

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

我们提出了一种慢病毒基因操纵和单一的嗅觉感官神经元轴突的可视化技术及其终端树枝状<em&gt;在体内</em&gt;。

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Research

JoVE Journal

Neuroscience

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

科研

神经科学

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录：神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...