Name: in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development
Uploaded: 2012-08-03T13:00:00
Description: Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

我们感谢孝彦松田博士提供pCAG-mCherry建设。陶博馆的支持由从Schweppe基金会事业发展奖“，从白厅基金会的资助。

1。提供制备（没有视频） <ol><li>青霉素/链霉素在1X PBS（PEN /链球菌）准备一个新鲜的1X稀释。不超过50毫升，是必要的。用0.22微米的注射器过滤器过滤。 </li><li>准备注射结合所需的质粒结构结构。到终体积10μL适当的化学计量学在的pCAG标志矢量我们把​​所有的结构。此外，14 pCAG-GFP或pCAG-mCherry应增加用于跟踪电效率和电穿孔细胞。我们用这个实验pCAG-mCherry。最后，在终浓度为0.05％，0...

<ol>
	<li>Wightman, R. M., Robinson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+changes+in+mesolimbic+dopamine+and+their+association+with+'reward'.">Transient changes in mesolimbic dopamine and their association with 'reward'.</a> J. Neurochem. 82, 721-735 (2002).</li><li>Kelley, A. E., Berridge, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroscience+of+natural+rewards:+relevance+to+addictive+drugs.">The neuroscience of natural rewards: relevance to addictive drugs.</a> J. Neurosci. 22, 3306-3311 (2002).</li><li>Moore, D. J., West, A. B., Dawson, V. L., Dawson, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathophysiology+of+Parkinson's+disease.">Molecular pathophysiology of Parkinson's disease.</a> Annu. Rev. Neurosci. 28, 57-87 (2005).</li><li>Dailly, E., Chenu, F., Renard, C. E., Bourin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine,+depression+and+antidepressants.">Dopamine, depression and antidepressants.</a> Fundam. Clin. Pharmacol. 18, 601-607 (2004).</li><li>Sesack, S. R., Carr, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+prefrontal+cortex+inputs+to+dopamine+cells:+implications+for+schizophrenia.">Selective prefrontal cortex inputs to dopamine cells: implications for schizophrenia.</a> Physiol. Behav. 77, 513-517 (2002).</li><li>Ang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+midbrain+dopaminergic+neuron+development.">Transcriptional control of midbrain dopaminergic neuron development.</a> Development. 133, 3499-3506 (2006).</li><li>Andersson, E. <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+intrinsic+determinants+of+midbrain+dopamine+neurons.">Identification of intrinsic determinants of midbrain dopamine neurons.</a> Cell. 124, 393-405 (2006).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Series+of+Normal+Stages+in+the+Development+of+the+Chick+Embryo.">A Series of Normal Stages in the Development of the Chick Embryo.</a> J. Morphol. 88, 49 (1951).</li><li>Bellairs, R., Osmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of chick development. , (2005).</li><li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">'Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1, 203-207 (1999).</li><li>Momose, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Muramatsu, T., Mizutani, Y., Ohmori, Y., Okumura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+nonviral+transfection+methods+for+foreign+gene+expression+in+early+chicken+embryos+in+ovo.">Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.</a> Biochem. Biophys. Res. Commun. 230, 376-380 (1997).</li><li>Nishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+in+vivo+gene+transfer+using+intraarterial+plasmid+DNA+injection+following+in+vivo+electroporation.">High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.</a> Cancer Res. 56, 1050-1055 (1996).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 16-22 (2004).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408 (2007).</li><li>Chesnutt, C., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid-based+short-hairpin+RNA+interference+in+the+chicken+embryo.">Plasmid-based short-hairpin RNA interference in the chicken embryo.</a> Genesis. 39, 73-78 (2004).</li></ol>

在鸡胚脑OVO电提供了一种低成本，快速的替代代转基因或基因敲除动物在中脑多巴胺神经元发育基因的功能进行的体内实验研究。使用短2毫米长L形的铂电极与胚胎脑特异的DNA注入是实现高效的兴趣在中脑多巴胺能神经元祖细胞的基因表达的关键。此外，使用正确的农户阶段11鸡胚是至关重要的。这是因为，第一，在农户阶段11，鸡脑的发展是不够先进等，前脑，中脑，后脑形...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>受精鸡蛋</td><td>查尔斯河实验室</td><td></td><td></td></tr><tr><td>鸡蛋孵化器</td><td> GQF制造</td><td> 1502运动员</td><td></td></tr><tr><td> A型肉毒毒素Electroporator </td><td> A型肉毒毒素哈佛仪器</td><td> ECM830 </td><td></td></tr><tr><td>电极</td><td> A型肉毒毒素哈佛仪器</td><td> 45-0162“L”形genetrodes </td><td> ECM830 electroporator </td></tr><tr><td>铂铱丝</td><td>阿法埃莎</td><td> ＃10056 </td><td> 0.5毫米直径 为了使2毫米长的电极</td></tr><tr><td>玻璃毛细管</td><td>世界精密仪器</td><td> TW100F-4 </td><td></td></tr><tr><td> microloader吸管提示</td><td> Eppendorf公司</td><td> 5242 956.003 </td><td></td></tr><tr><td>墨汁</td><td>斯台普斯</td><td></td><td>过滤器在使用前20％ </td></tr><tr><td>微量注射器</td><td>帕克自动化， Parker Hannifin公司合作</td><td> picospritzer三</td><td></td></tr><tr><td>荧光解剖显微镜</td><td>徕卡</td><td> MZ16F </td><td></td></tr><tr><td>微管拔</td><td>萨特仪器</td><td>的D-97 </td><td></td></tr></tbody></table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

多巴胺能神经元位于中腹脑控制的运动，情绪化的行为，和奖励机制1-3。腹侧中脑多巴胺神经元功能障碍是帕金森氏症，精神分裂症，抑郁症，老年痴呆症1-5牵连。因此，研究中脑多巴胺神经元分化的调节不能只提供重要的洞察到调节脑发育和神经祖命运规范机制，而且还有助于开发治疗多种人类神经系统疾病的新的治疗策略。 多巴胺神经元分化从排队心室胚胎腹侧中脑区的神经祖细胞。神经祖细胞的发展，控制基因表达方案6,7。在这里，我们报告技术利用电汉堡汉密尔顿（HH）阶段11脑基因表达（THirteen体节，42小时）鸡胚8,9。允许在特定的萌芽阶段的实验操作方便，在后来的发展时间点10-13决定的影响，的鸡胚外部发展。鸡胚早期阶段，13（19体节，48小时）超过HH神经管组成的具有多能分化成不同类型的细胞神经系统的神经祖细胞。的pCAG载体，其中包含CMV启动子和小鸡β-actin的增强，使强大的标志或其他抗原标记的鸡胚神经管14结构表达。在这份报告中，我们强调特别措施，以实现区域限制的基因表达，在胚胎中脑多巴胺能神经元祖细胞，包括如何注入DNA构建专门的胚胎中脑区域，以及如何针对小定制电极电。分析C希克在稍后阶段的脑体内质粒载体介导的增益功能和亏损的功能研究脑发展系统提供一个很好的。实验系统的改造，可以延长该法执行的命运映射分析和研究基因表达调控神经系统的其他部分。

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

评估的功能和在发展中脑多巴胺能神经元的基因调控，我们描述了一种方法，涉及<em&gt;在OVO</em&gt;鸡胚腹侧中脑多巴胺能神经元祖细胞质粒DNA结构的电。这种技术可以用来实现感兴趣的基因的高效表达，以研究脑发展和多巴胺神经元分化的不同方面。

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Research

JoVE Journal

Neuroscience

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

In ovo Expression of MicroRNA in Ventral Chick Midbrain

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Studying Brain Function in Children Using Magnetoencephalography

脑磁图在儿童脑功能研究中的应用

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...