Name: in ovo electroporation in the chicken auditory brainstem
Uploaded: 2017-06-09T13:00:00
Description: Watch this Scientific Journal Video about In Ovo Electroporation in the Chicken Auditory Brainstem at JoVE.com

我们要感谢Drs。 Leslayann Schecterson，Yuan Wang，Andres Barria和Ximena Optiz-Araya太太，为协议设置和提供质粒提供初步帮助。这项工作得到NIH / NIDCD授权DC013841（JTS）的支持。

所有程序均经西北大学机构动物护理和使用委员会批准，并按照国家卫生研究院“实验动物护理和使用指南”进行。 蛋处理<ol><li>从当地的供应商处购买受精卵。在孵育前不要超过5天，将冰箱中的鸡蛋在13°C储存。 1周后胚胎活力明显下降。 </li><li>用70％乙醇清洗每个鸡蛋，然后放入培养箱中。 </li><li>在温度设定在38°C〜50％湿度的孵化器中，在其侧面设置1-2打鸡蛋。这确保...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+architecture+in+nucleus+magnocellularis+of+the+chicken+auditory+system+with+observations+on+nucleus+laminaris:+a+light+and+electron+microscope+study.">Neuronal architecture in nucleus magnocellularis of the chicken auditory system with observations on nucleus laminaris: a light and electron microscope study.</a> Neuroscience. 7 (4), 809-836 (1982).</li><li>Matsui, R., Tanabe, Y., Watanabe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+adeno-associated+virus+vector+efficiently+transduces+neurons+in+the+embryonic+and+post-embryonic+chicken+brain.">Avian adeno-associated virus vector efficiently transduces neurons in the embryonic and post-embryonic chicken brain.</a> PLoS One. 7 (11), e48730 (2012).</li><li>Koppl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+nerve+terminals+in+the+cochlear+nucleus+magnocellularis:+differences+between+low+and+high+frequencies.">Auditory nerve terminals in the cochlear nucleus magnocellularis: differences between low and high frequencies.</a> J Comp Neurol. 339 (3), 438-446 (1994).</li><li>Hyson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+interaural+time+differences+in+the+chick+brain+stem.">The analysis of interaural time differences in the chick brain stem.</a> Physiol Behav. 86 (3), 297-305 (2005).</li><li>Jones, T. A., Jones, S. M., Paggett, 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=Emergence+of+hearing+in+the+chicken+embryo.">Emergence of hearing in the chicken embryo.</a> J Neurophysiol. 96 (1), 128-141 (2006).</li><li>Saunders, J. C., Coles, R. B., Gates, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+auditory+evoked+responses+in+the+cochlea+and+cochlear+nuclei+of+the+chick.">The development of auditory evoked responses in the cochlea and cochlear nuclei of the chick.</a> Brain Res. 63, 59-74 (1973).</li><li>Woolf, N. K., Ryan, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+auditory+function+in+the+cochlea+of+the+mongolian+gerbil.">The development of auditory function in the cochlea of the mongolian gerbil.</a> Hear Res. 13 (3), 277-283 (1984).</li><li>Walsh, E. 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ovo电穿孔是一种表达或敲低感兴趣的基因以分析体内基因功能的方法8,9 。在鸡胚中，将质粒编码的基因表达到不同的听觉脑干区域中是一种创新的方法。为了确保最佳表达，需要几个关键步骤。首先，只注射其囊肿清晰可见的胚胎。如果卵巢不可见，则胚胎不能在电穿孔的正确发育阶段。第二，较小的移液管尖端使得更容易穿透神经管并导致更少的组织损伤。然?...

声音的快速神经编码对于正常的听觉功能至关重要。这些包括声音定位能力1 ，噪音识别2中的语音，以及其他与行为相关的通信信号3的理解。鸟类和哺乳动物的听觉脑干中的类似神经元是高度专门用于快速神经编码的。其中包括鸡核细胞核（NM），核心层流（NL）及其哺乳动物类似物，前内耳蜗核（AVCN）和内侧上橄榄（MSO） 5 。然而，在听觉脑干中，调节快速神经编码的发育机制知之甚少。因此，研究负责快速神经编码的具体基因是为了更好地了解其在au中的表达，调控和功能是有利的发展。 发展中的鸡胚是研究听觉系统发展基础生物学问题的有效和成熟的研究工具。最近的分子进展已经在发展中的鸡胚中通过表达或敲低感兴趣的基因来解决这些生物学问题，以分析体内基因功能8,9 。研究特定基因的调节作用是理解与听觉缺陷相关的病理学的重大进步。在这里，我们将质子编码基因的ovo电穿孔进入鸡听觉脑干，其中声音的快速神经编码发生10 。通过靶向与菱形体5和6相关的听觉神经祖细胞区域11,12（R5 /R6），我们显示了NM和NL中质粒转染的空间控制。此外，我们通过采用tet-on向量系统显示表达的时间调节。这是在多西环素（Dox） 8存在下表达感兴趣的基因的药物诱导性程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fertilized white leghorn chicken eggs</td>
			<td>Sunnyside Inc. (Beaver Dam, WI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td>Picospritzer III, single or dual channel</td>
		</tr>
		<tr>
			<td>Current/voltage stimulator</td>
			<td>Grass Technologies</td>
			<td>SD9</td>
			<td>SD9</td>
		</tr>
		<tr>
			<td>Microfil syringe needles</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td>28 Gauge, 67 mm Long, (Pack of 5)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Warner Instruments</td>
			<td>64-1280</td>
			<td>MP Series: Non-Electrical Pressure Applications</td>
		</tr>
		<tr>
			<td>Stimulating microelectrode</td>
			<td>FHC</td>
			<td>PBSA1075</td>
			<td>PBSA1075</td>
		</tr>
		<tr>
			<td>Air tank/regulator</td>
			<td>NU Laboratory Services</td>
			<td></td>
			<td>Air dry 300 CF</td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Sigma Aldrich</td>
			<td>F7258-25G</td>
			<td>F7258-25G</td>
		</tr>
		<tr>
			<td>Clear plastic tape</td>
			<td>Scotch</td>
			<td>191</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline hyclate</td>
			<td>Sigma Aldrich</td>
			<td>D9891-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg refrigerator</td>
			<td>Vissani Wine Refrigerator</td>
			<td></td>
			<td>13.3-16.1&#176; C (56-61&#176; F)</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hova-Bator</td>
			<td></td>
			<td>37.8&#176; C (100&#176; F), ~50% humidity</td>
		</tr>
		<tr>
			<td>Dissection scope</td>
			<td>Zeiss</td>
			<td>4.35E+15</td>
			<td>SteREO Discovery, V8 Microscope, 50.4X</td>
		</tr>
		<tr>
			<td>Cold-light source</td>
			<td>Zeiss</td>
			<td>4.36E+15</td>
			<td>CL6000 LED</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Narishige Japan</td>
			<td>Model: MM-3</td>
			<td>2 Micromanipulators</td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>Sutter Instrument</td>
			<td>BF150-86-10</td>
			<td>Thick-walled borosilicate (dimensions)</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td></td>
			<td>1 mL, 3 mL</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>BD Precision Glide&#160;</td>
			<td></td>
			<td>27 G x 1 1/4, 19 G x 1 1/2</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Stoelting</td>
			<td></td>
			<td>No. 5 Super Fine Dumont</td>
		</tr>
		<tr>
			<td>Egg holder</td>
			<td>Custom Made</td>
			<td></td>
			<td>Clay base works as well</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td></td>
			<td>Model P-97</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Ultra Cruz</td>
			<td>sc-358811</td>
			<td>PVDF 0.22 &#956;m</td>
		</tr>
	</tbody>
</table>

in ovo electroporation in the chicken auditory brainstem

电穿孔是将感兴趣的基因引入生物相关生物体如鸡胚的方法。长期以来，鸡胚是研究听觉系统发育基本生物学功能的有效研究模式。最近，鸡胚在研究与听力相关的基因表达，调节和功能方面已经变得特别有价值。 在ovo电穿孔可用于瞄准听觉脑干区域负责高度专业化的听觉功能。这些区域包括鸡核细胞（NM）和核层（NL）。 NM和NL神经元来自菱形体5和6的不同前体（R5 / R6）。在这里，我们介绍质粒编码基因的ovo电穿孔研究这些区域的基因相关性质。我们显示一种促进功能表型增益或丧失的基因表达空间和时间控制的方法ES。通过靶向与R5 / R6相关的听觉神经祖细胞区域，我们显示在NM和NL中的质粒转染。通过采用tet-on载体系统可以实现基因表达的时间调控。这是在多西环素（Dox）存在下表达感兴趣的基因的药物诱导性程序。 卵内电穿孔技术 - 与生物化学，药理学和/或体内功能测定一起提供了一种创新的方法来研究听觉神经元发育和相关的病理生理现象。

我们在这里显示， 在ovo电穿孔允许在正常发展的生物系统中的基因表达。质粒编码的基因被重点注射到覆盖R5 / R6的神经管中。 图1A中示出了相对于重要的解剖学标记的电极和移液器放置的示意性示例。在电穿孔后24小时证实质粒注射的正确位置， 如图1B所示 。将质粒靶向注射到R5 / R6上的神经管中，允许在特定的听觉脑干区域（即NM和NL <sup...

Watch this Scientific Journal Video about In Ovo Electroporation in the Chicken Auditory Brainstem at JoVE.com

In Ovo Electroporation in the Chicken Auditory Brainstem

鸟类和哺乳动物的听觉脑干神经元专门用于快速神经编码，这是正常听力功能的基本过程。这些神经元来自胚胎后脑的不同前体。我们提出利用电穿孔技术在鸡胚后脑中表达基因以研究听觉发育过程中的基因功能。

在奥运电击鸡听觉脑干中

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Research

JoVE Journal

Developmental Biology

In Ovo Electroporation in the Chicken Auditory Brainstem

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

鸡胚全视网膜外植体电穿孔和化学试剂处理

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

一本小说<em&gt;防爆大毛</em&gt;带技术来改变心内血流动力学在鸡胚胎系统

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Electroporation-mediated RNA Interference Method in Odonata

奥多纳塔电介质介质RNA干扰法

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...