Name: genetic study of axon regeneration with cultured adult dorsal root ganglion neurons
Uploaded: 2012-08-17T12:00:00
Description: Watch this Scientific Journal Video about Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons at JoVE.com

FZ型从NIH（R01NS064288）和克雷格H.尼尔森基金会的赠款，以支持这项工作。

1。盖玻片，培养基，消化酶的制备<ol><li> 12毫米的圆形＃1盖玻片用于神经元的文化。清洁的盖玻片，10％HCL一夜之间随后用蒸馏水和去离子水超声波清洗3次（20分钟/次）。清洗盖玻片存储在70％乙醇以供将来使用。在每次实验中，盖玻片是空气干燥，并放置到培养皿。 </li><li>涂层干燥的盖玻片，100μL涂层溶液含有100微克/毫升聚-D-赖氨酸和10微克/毫升层粘连工作的解决方案添加到每个盖玻片转?...

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	<li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat. Rev. Neurosci. 7, 617-627 (2006).</li><li>Liu, K., Tedeschi, A., Park, K. K., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Intrinsic+Mechanisms+of+Axon+Regeneration.">Neuronal Intrinsic Mechanisms of Axon Regeneration.</a> Annu. Rev. Neurosci. , (2010).</li><li>Zhou, F. Q., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+control+of+developmental+and+regenerative+axon+growth.">Intracellular control of developmental and regenerative axon growth.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 361, 1575-1592 (2006).</li><li>Neumann, S., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+dorsal+column+fibers+into+and+beyond+the+lesion+site+following+adult+spinal+cord+injury.">Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.</a> Neuron. 23, 83-91 (1999).</li><li>Liu, R. Y., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+signaling+pathways+mediate+regenerative+versus+developmental+sensory+axon+growth.">Different signaling pathways mediate regenerative versus developmental sensory axon growth.</a> J. Neurosci. 21, RC164 (2001).</li><li>Zhou, F. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+support+regenerative+axon+assembly+over+CSPGs+by+an+ECM-integrin-independent+mechanism.">Neurotrophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism.</a> J. Cell Sci. 119, 2787-2796 (2006).</li><li>Zou, H., Ho, C., Wong, K., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axotomy-induced+Smad1+activation+promotes+axonal+growth+in+adult+sensory+neurons.">Axotomy-induced Smad1 activation promotes axonal growth in adult sensory neurons.</a> J. Neurosci. 29, 7116-7123 (2009).</li><li>Zhou, F. Q., Zhou, J., Dedhar, S., Wu, Y. H., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF-induced+axon+growth+is+mediated+by+localized+inactivation+of+GSK-3beta+and+functions+of+the+microtubule+plus+end+binding+protein+APC.">NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC.</a> Neuron. 42, 897-912 (2004).</li><li>Saijilafu, E. M., Hur, F. Q., Zhou, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+axon+regeneration+via+in+vivo+electroporation+of+adult+mouse+sensory+neurons.">Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons.</a> Nat. Commun. 2, 543-54 (2011).</li><li>Costigan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicate+high-density+rat+genome+oligonucleotide+microarrays+reveal+hundreds+of+regulated+genes+in+the+dorsal+root+ganglion+after+peripheral+nerve+injury.">Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.</a> BMC Neurosci. 3, 16 (2002).</li><li>Xiao, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+gene+expression+profile+of+dorsal+root+ganglion+in+the+rat+peripheral+axotomy+model+of+neuropathic+pain.">Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 8360-8365 (2002).</li><li>Michaelevski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+to+transcription+networks+in+the+neuronal+retrograde+injury+response.">Signaling to transcription networks in the neuronal retrograde injury response.</a> Sci. Signal. 3, ra53 (2010).</li></ol>

成年DRG神经元在体内和体外的周围神经损伤后轴突再生强劲，从而提供了一个有用的系统研究在成年动物的神经轴突再生。成年DRG神经元在体外培养成为一种广泛使用的方法来研究的分子机制轴突再生的调节。 在体外培养成年小鼠DRG神经元的过程中所提出的在这里可以迅速和有效的再生轴突生长的遗传研究。再悬浮和再镀过程是亏损的功能研究特别有用，因为它允许轴...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>的MEM </td><td> Invitrogen公司</td><td> 11090-081 </td></tr><tr><td>聚-D-赖氨酸氢溴酸</td><td> Sigma-Aldrich公司</td><td> P6407 </td></tr><tr><td>层粘连蛋白</td><td> Invitrogen公司</td><td> 23017-015 </td></tr><tr><td> 5 - 氟-2-脱氧</td><td> Sigma-Aldrich公司</td><td> F0503 </td></tr><tr><td>尿苷</td><td> Sigma-Aldrich公司</td><td> U3003 </td></tr><tr><td>胶原酶一个</td><td>罗氏公司</td><td> 10103578001 </td></tr><tr><td> TrypLE快递</td><td> Invitrogen公司</td><td> 12604-013 </td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 10270-098 </td></tr><tr><td>青霉素，链霉素（100X） </td><td> Invitrogen公司</td><td> 15140-122</td></tr><tr><td> GlutaMAX我（100X） </td><td> Invitrogen公司</td><td> 35050-038 </td></tr><tr><td>盖玻片（＃1） </td><td>电子显微镜科学</td><td> 72196-12 </td></tr><tr><td> 24孔细胞培养板</td><td>碧迪</td><td> 35-3047 </td></tr><tr><td> 1×PBS </td><td>敏达</td><td> 21-040-CV </td></tr><tr><td>无菌蒸馏水和去离子水</td><td>敏达</td><td> 25-055-CV </td></tr><tr><td>小鼠神经元nucleofector和电套件</td><td>龙沙</td><td> VPG-1001 </td></tr><tr><td>对c-Jun的ON TARGETplus的siRNA </td><td> Dharmacon的</td><td> L - 043776 </td></tr><tr><td>反 - βIII微管蛋白抗体（Tuj的1） </td><td> Covance公司</td><td> MMS-435P </td></tr><tr><td>延长黄金抗淬灭安装解决方案</td><td> Invitrogen公司</td><td> P36930 </td></tr></TBODY&gt; </table>

genetic study of axon regeneration with cultured adult dorsal root ganglion neurons

这是众所周知的，成熟的神经元在中枢神经系统（CNS）不能再生它们的轴突受伤后，由于减少的内在能力，以支持在1,2成熟的中枢神经系统轴突生长和充满敌意的环境。相比之下，在成熟的神经元周围神经系统（PNS）的再生容易受伤后3。成年猫背根神经节（DRG）神经元是众所周知的有力周围神经损伤后再生。每背根神经节神经元增长1轴突从胞体，分支成两个轴突分支：外围分支支配外围目标和中央部门，延伸到脊髓。损伤的背根神经节的末梢神经轴突大量的轴突再生的结果，而中央在脊髓轴突再生的受伤后不佳。但是，如果发生周围性轴索损伤脊髓损伤（这个过程被称为空调病变），中央轴突再生前是greatlŸ提高4。此外，中央DRG神经元轴突共享降皮质脊髓轴突充满敌意的环境。在一起，这是假设，控制成年DRG神经元的轴突再生的分子机制可以利用，以提高中枢神经系统轴突再生。因此，现在广泛使用的成人DRG神经元作为一个模型系统研究再生轴突生长5-7。 在这里，我们描述了一个成人，DRG神经元培养轴突再生的基因研究在体外可以使用的方法。在这个模型成年DRG神经元的基因通过电穿孔介导的基因转染6,8操纵。转染神经元的DNA质粒或SI / shRNA的，这种方法能够使增益和亏损的功能试验，以调查任何在成年DRG神经元的轴突生长的基因利益的作用。当神经元与SI / shRNA的转，有针对性的内源性蛋白通常耗尽后3-4天，在文化，在这期间，强大的轴突生长已经发生，减少损失的功能研究有效。为了解决这个问题，这里介绍的方法，包括一个染后的再悬浮和再镀步，使轴突从神经元的目标蛋白质的情况下重新长出。最后，我们利用体外模型研究轴突再生相关基因c-Jun的作用，在调解成年DRG神经元的轴突生长提供了一个例子。

Watch this Scientific Journal Video about Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons at JoVE.com

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

一个<em&gt;在体外</em用培养的成年小鼠背根神经节神经元的轴突再生的遗传研究模型描述。方法包括re-suspension/re-plating的步骤，允许进行遗传操作的神经元的轴突重新生长。这种做法是轴突再生功能丧失使用RNAi为基础的蛋白质击倒的研究特别有用。

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Research

JoVE Journal

Neuroscience

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

背根神经节神经元和区分脂肪干细胞：一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

科研

神经科学

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

在癌神经侵袭的体外模型：背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Laminotomy 对猪腰背根神经节通路和注射的治疗

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

使用初级培养的希波坎帕神经元来研究Axon初始片段的组装

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...