Name: in ovo and ex ovo methods to study avian inner ear development
Uploaded: 2022-06-16T15:00:00
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我们非常感谢NCBS，TIFR，Infosys-TIFR前沿研究基金，DST-SERB和皇家国家聋人研究所的支持。我们要感谢班加罗尔赫萨拉加塔的中央家禽发展组织和培训学院。我们感谢CIFF和EM设施和NCBS的实验室支持。我们感谢Yoshiko Takahashi和Koichi Kawakami的Tol2-eGFP和T2TP构建体，以及Guy Richardson的HCA和G19 Pcdh15抗体。我们感谢 Earlab 成员对该协议的持续支持和宝贵反馈。

涉及受精鸡蛋和未孵化胚胎的采购，培养和使用的方案由卡纳塔克邦班加罗尔国家生物科学中心的机构动物伦理委员会批准。
1. 在 卵电穿孔中雏 鸡听觉前体
<ol><li>用于CRISPR/Cas9基因敲除的sgRNA设计和克隆<ol><li>为了创建基因敲除，设计引导RNA破坏基因的外显子区域，最好靠近编码区域的5'端。</li>
		<li>使用网络工具CRISPOR23选择潜在的向导...

<ol>
	<li>Sai, X., Ladher, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+steps+in+inner+ear+development:+induction+and+morphogenesis+of+the+otic+placode.">Early steps in inner ear development: induction and morphogenesis of the otic placode.</a> Frontiers in Pharmacology. 6, 19 (2015).</li><li>Groves, A. K., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+sound+in+space:+the+regulation+of+inner+ear+patterning.">Shaping sound in space: the regulation of inner ear patterning.</a> Development. 139 (2), 245-257 (2012).</li><li>Driver, E. C., Kelley, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+filaments,+stereocilia,+and+hair+cells+of+the+bird+cochlea.+IV.+How+the+actin+filaments+become+organized+in+developing+stereocilia+and+in+the+cuticular+plate.">Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.</a> Developmental Biology. 116 (1), 119-129 (1986).</li><li>Oesterle, E. C., Tsue, T. T., Reh, T. A., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-cell+regeneration+in+organ+cultures+of+the+postnatal+chicken+inner+ear.">Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.</a> Hearing Research. 70 (1), 85-108 (1993).</li><li>Honda, A., Freeman, S. D., Sai, X., Ladher, R. K., O'Neill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+labyrinth:+culture+of+the+chicken+inner+ear.">From placode to labyrinth: culture of the chicken inner ear.</a> Methods. 66 (3), 447-453 (2014).</li><li>Matsunaga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+supporting+cell+activation+for+hair+cell+regeneration+in+the+avian+auditory+epithelium:+an+explant+culture+model.">Initiation of supporting cell activation for hair cell regeneration in the avian auditory epithelium: an explant culture model.</a> Frontiers in Cellular Neuroscience. 14, 583994 (2020).</li><li>Concordet, J. 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雏鸡是实验室可以用来研究内耳的模式生物的一种经济高效且方便的补充。这里描述的方法在我们的实验室中经常使用，并补充了正在进行的哺乳动物内耳研究。 在卵子中 ，电穿孔用于将遗传操作引入鸡基因组。电穿孔还可用于引入编码靶向特定细胞器或亚细胞结构的荧光蛋白的构建体35，36。虽然这是一个简单的程序，但在 卵子中 操纵...

所有脊椎动物的内耳都来源于一个简单的上皮，称为耳基板1，2。这将产生所有必要的结构元素和细胞类型，以转导与听觉和平衡感知相关的机械感觉信息。毛细胞（HCs）是内耳的纤毛传感器，被支持细胞（SCs）包围。HC通过第八颅神经的神经元将信息传递给听觉后脑。这些也是从耳基板3产生的。声音的主要转导是在听觉HC的顶端表面通过机械敏感的毛束4实现的。这是通过修饰的基于肌动蛋白的突起（称为立体纤毛）介导的，这些突起以分级的阶梯模式排列5。此外，一种改良的初级纤毛，称为 kinocilium，组织毛束形成，并与最高的立体纤毛6，7，8 行相邻。立体纤毛的结构对于将源自声能的机械刺激转换为电神经信号的作用至关重要9。衰老、感染、耳声创伤或耳毒性休克对听觉HC的损害可导致部分或完全听力损失，在哺乳动物中，这是不可逆转的10。
已经提出了可能修复这种损伤的细胞替代疗法11，12。这项研究的方法是了解哺乳动物毛细胞的正常发育，并询问是否可以在内耳内可能存在的祖细胞样细胞中重新启动发育程序13。第二种方法是将目光投向哺乳动物之外，寻找非哺乳动物脊椎动物，其中听觉毛细胞发生强劲再生，例如鸟类14，15。在鸟类中，毛细胞再生主要通过支持细胞去分化为祖细胞样状态发生，然后进行不对称有丝分裂以产生毛细胞和支持细胞16。此外，还观察到支持细胞的直接分化以产生毛细胞17。
虽然鸟类听觉发育的机制确实与哺乳动物有显着相似之处，但存在差异18。雏鸡血压的HC和SC分化从胚胎第7天（E）开始就很明显，随着时间的推移变得更加明显。通过E12，可以看到图案良好且极化良好的基底（BP），通过E17可以看到发育良好的毛细胞19。这些时间点为分化、图案化和极性以及毛细胞成熟的机制提供了窗口。了解这些机制是保守的还是发散的很重要，因为它们提供了对机械感觉毛细胞起源的深层同源性的见解。
在这里，我们展示了在胚胎早期和晚期进行的一系列技术，以研究细胞过程，例如内耳器官发育过程中的增殖，命运规范，分化，模式化和维持。这补充了了解外植体培养中内耳发育的其他协议20，21，22。我们首先讨论使用卵电穿孔将外源性DNA或RNA引入E3.5耳囊内的BP前体中。尽管遗传操作可以提供有价值的见解，但由此产生的表型可能是多效性的，因此是混淆的。在后期的内耳发育中尤其如此，其中细胞骨架重塑等基本过程在细胞分裂、组织形态发生和细胞特化中起着多种作用。我们提出了培养外植体的药理学抑制方案，其在控制剂量和治疗时间和持续时间方面具有优势，提供了发育机制的精确时空操作。
根据小抑制剂的治疗持续时间，可以使用不同的器官培养方法。在这里，我们展示了两种器官培养方法，可以深入了解上皮形态发生和细胞特化。一种使用胶原蛋白作为基质来培养耳蜗导管的 3D 培养方法可实现发育中的血压的稳健培养和实时可视化。为了了解立体纤毛的形成，我们提出了一种膜培养方法，使得上皮组织在刚性基质上培养，使肌动蛋白突起能够自由生长。这两种方法都允许下游处理，例如活细胞成像，免疫组织化学，扫描电子显微镜（SEM），细胞记录等。这些技术为有效使用雏鸡作为模型系统来理解和操纵鸟类听觉上皮的发育、成熟和再生提供了路线图。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A22287</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. HiFi Cas9 Nuclease V3</td>
			<td>Integrated DNA Technologies</td>
			<td>1081061</td>
			<td>High fidelity Cas9 protein</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam</td>
			<td>ab290</td>
			<td>Rabbit polyclonal to GFP</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q12135</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, rat tail</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1048301</td>
			<td></td>
		</tr>
		<tr>
			<td>Critical Point Dryer Leica EM CPD300</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CUY-21 Electroporator</td>
			<td>Nepagene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>DM5000B Widefield Microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield</td>
			<td>Sigma-Aldrich</td>
			<td>F6182</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOVIEW 3000 Laser Scanning Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25 %)</td>
			<td>Sigma-Aldrich</td>
			<td>340855</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11032</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum Sterile filtered</td>
			<td>HiMedia</td>
			<td>RM10701</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM980 Airyscan Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 &#181;m</td>
			<td>Sigma-Aldrich</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>MVX10 Stereo Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MYO7A antibody</td>
			<td>DSHB</td>
			<td>138-1</td>
			<td>Mouse monoclonal to Unconventional myosin-VIIa</td>
		</tr>
		<tr>
			<td>MZ16 Dissecting microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes Scissors, 14cm (5.5&#39;&#39;)</td>
			<td>World Precision Instruments</td>
			<td>501237</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide (4%)</td>
			<td>Sigma-Aldrich</td>
			<td>75632</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-10 Puller</td>
			<td>Narishige</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pcU6_1sgRNA</td>
			<td>Addgene</td>
			<td>92395</td>
			<td>Mini vector with modified chicken U6 promoter</td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>SMZ1500 Dissecting microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate Buffer, 0.2M</td>
			<td>Electron Microscopy Sciences</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>HiMedia</td>
			<td>GRM853</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputtre Coater K550X</td>
			<td>Emitech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Glass Capillaries 3 in, OD 1.0 mm, No Filament</td>
			<td>World Precision Instruments</td>
			<td>1B100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr>
			<td>The MERLIN Compact VP</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Alfa Aesar</td>
			<td>L01205</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
	</tbody>
</table>

in ovo and ex ovo methods to study avian inner ear development

内耳感知声音并使用耳蜗和前庭保持平衡。它通过使用称为毛细胞的专用机械感觉细胞类型来做到这一点。内耳的基础研究使人们深入了解了毛细胞的功能，以及失调如何导致听力损失和眩晕。在这项研究中，鼠标一直是杰出的模型系统。然而，像所有哺乳动物一样，小鼠已经失去了替代毛细胞的能力。因此，当试图了解恢复内耳功能的细胞疗法时，对其他脊椎动物物种的补充研究可以提供进一步的见解。鸟类的听觉上皮，基底（BP），是由机械感觉毛细胞（HC）组成的上皮片，由支持细胞（SC）插入。虽然基底和哺乳动物耳蜗的解剖结构不同，但内耳发育和听力的分子机制相似。这使得基底不仅成为比较研究的有用系统，而且有助于了解再生。在这里，我们描述了鸡内耳的解剖和操作技术。该技术显示了遗传和小分子抑制方法，为研究内耳发育的分子机制提供了有效的工具。在本文中， 我们讨论了使用 CRIPSR-Cas9缺失对基底进行遗传扰动的卵电穿孔技术，然后解剖基底。我们还展示了BP器官培养和培养基质的最佳使用，以观察上皮和毛细胞的发育。

在电穿孔设置中，电极定位可以在转染领域发挥作用。正极放置在蛋黄下方，负极放置在胚胎上方（图1A）。这导致大部分内耳和两个前庭器官（图1B）和听基底（图1C，D）的GFP表达较高，证实了转染。
在评估CRISPR敲低的表型时，我们设计了引导RNA到毛细胞转录因子Atonal homolog1（Atoh1）。Atoh1的小?...

Watch this Scientific Journal Video about In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development at JoVE.com

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

雏鸡是一种具有成本效益、可及且广泛可用的模式生物，可用于各种研究。在这里，详细介绍了一系列协议，以了解鸟类内耳发育和再生的分子机制。

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

年龄、感染和耳毒性药物会导致内耳毛细胞退化，从而导致听力损失。在非哺乳动物脊椎动物中，如小鸡，毛细胞可以再生。这里描述的技术利用了在雏鸡胚胎上工作的成本效益，获得胚胎的便利性以及组织外植体的良好外显。了解鸟类内耳毛的发育和再生可以为听力损失和潜在疗法提供重要的见解，外植体培养对于评估各种药物的耳毒性作用非常重要。在开始实验之前，采购新鲜产下的鸡蛋，并用70%乙醇清洁它们以防止污染。将种蛋在 37 至 38 摄氏度、湿度为 45% 的条件下孵化 3.5 至 4 天。孵化后，通过将鸡蛋侧放五分钟，将胚胎重新定位到蛋黄的顶部。然后，用镊子在鸡蛋的顶部和钝端开一个小孔，让 21 号针穿过。使用5毫升注射器和21号针头，从鸡蛋钝端的孔中取出两毫升白蛋白。用透明胶带覆盖钝端的孔。为了使鸡蛋窗口将透明胶带贴在蛋壳的顶部。使用垂直移液器拉拔器从标准玻璃毛细管中握住显微注射的针头。拉动后，使用细镊子折断毛细管尖端，以获得约50微米的尖端直径和锥形末端。对于基因敲除实验，将三种构建体，即引导质粒，pcU6.1sgRNA，T2KeGFP，T2TP与1μg的SP Cas9蛋白，30%蔗糖和0.1%快速绿色染料以一比一的比例混合，最终体积为10微升。在电移多个质粒时，确保DNA的最终浓度至少为每微升4微克。在弹簧弓剪刀的帮助下，在两厘米长和1.5厘米宽的窗户周围切开，露出胚胎。然后用镊子打开覆盖在胚胎上的绒毛膜，允许进入胚胎。注入约200纳升体积的DNA溶液混合物以填充耳囊泡。为了确定引导RNA效率，请进行T7核酸内切酶测定。向胚胎中添加0.719%盐水滴剂以降低电阻，并防止过热。接下来，将正极穿过鸡蛋钝侧的孔，并操纵电极使其位于蛋黄下方。将负极放在填充的耳囊上。要通过电穿孔将质粒转染到耳囊泡中，请使用方形脉冲发生器，并施加五个 25 伏脉冲，每个脉冲 100 毫秒，间隔 50 毫秒。根据单个电穿孔设置凭经验确定条件。电穿孔后，通过添加几滴0.719%盐水来水合胚胎。用透明胶带重新密封鸡蛋，然后返回 37 至 38 摄氏度的加湿孵化器进行进一步孵化。使用70%乙醇和加热对手术台，显微镜载物台和周围区域进行消毒，或酒精对微解剖设备进行消毒，包括最小弹簧弓剪刀，微型刮匙和两对细钳。准备解剖板，包括带有黑色硅胶底座的玻璃培养皿，90毫米塑料培养皿和60毫米培养皿。保持冷藏的PBS或汉克斯平衡盐溶液（也称为HBSS）准备好进行解剖。轻轻地将鸡蛋打入 90 毫米培养皿中。然后识别雏鸡的外耳，并将头部转移到装有冰冷PBS的60毫米培养皿中。接下来，将喙的顶部朝内定向胚胎，并使用五号镊子之一握住喙。用第二个五号镊子挖出眼睛。然后，从喙部到尾部，沿着头骨的中线切开，挖出大脑。添加更多冰冷的PBS或HBSS，并将拉根的两个耳石定位为耳蜗管末端的闪亮结构，靠近中线。为了隔离两个内耳，在两个拉根之间切开，并在该区域的上方和下方切开。然后在倾斜的照明下可视化内耳的轮廓，并去除多余的组织和前庭。将分离的耳蜗转移到带有冰冷PBS的黑色硅胶底板上。使用五号镊子剥离软骨耳蜗囊以获得耳蜗管。然后找到耳蜗管的起伏层或被盖，并使用55号镊子将其取出，露出基底或BP。接下来，去除盖膜以使用55号镊子暴细胞和支持细胞。对于基底的膜培养，取一个六孔组织培养板，每孔布置一个培养膜插入物。在200微升移液管中吸取一些培养基，然后用一个X HBSS缓冲液吸出解剖的基底外植体。将外植体转移到膜上，并将其定向，使基底朝上，并且从顶部可以看到毛发和支持细胞。一旦外植体定位，从培养膜表面缓慢吸出HBSS缓冲液。在此过程中，外植体将附着在培养膜上。通过在膜插入物和孔壁之间添加 1.2 毫升 dulbecco 的改良鹰培养基或 DMEM 培养基来填充六孔板的孔。为了制备耳蜗管的胶原蛋白培养物，将三滴新鲜制备的胶原蛋白混合物加入四孔板的每个孔中，并将解剖的耳蜗管转移到每个胶原蛋白滴中。将板在37摄氏度和5%二氧化碳下孵育10分钟以固化胶原蛋白基质。对于培养物的小分子处理，用补充有药理调节剂（如青霉素）的700微升培养基替换培养基，并如前所述孵育平板。每天更换50%的培养基。在适当的孵育时间后，取出培养基，并使用外植体进行下游测定。在电穿孔设置中，正确的电极定位导致两个前庭器官在内耳中更高的GFP表达，以及确认转染的听基底。CRISPR Cas9介导内耳Atoh1基因敲除，导致毛细胞流失。Atoh1基因敲除通过卵电穿孔，然后孵育直到E10显示与空质粒对照相比，毛细胞发育减少。虽然电穿孔是马赛克，但对照电穿孔细胞能够形成毛细胞。在Atoh1 gRNA电穿孔样品中，绿色荧光蛋白阳性细胞从未显示出毛细胞发育的标志物。基底在3D基质中的器官培养，例如胶原蛋白和带有毛细胞抗原抗体的染色剂，提供了长达五天的组织形态的出色保存。在这些培养条件下维持毛细胞和支持细胞的组织。膜上的器官培养物可以维持长达五天，同时保持毛束的完整性。这可以在代表性图像中看到，通过尖端链接蛋白原钙粘蛋白15的定位。为了研究毛束的发展，更高分辨率的成像，如超分辨率显微镜或扫描电子显微镜可以提供更多信息。在整个过程中应保持无菌条件。电泳时，确保胚胎不会变干。滴定药理学抑制剂时，可以降低浓度以克服任何致死性。胚胎和外植体都可以用于成像实验。通过执行实时视频微观范围或静态光共聚焦电子显微镜，如协议所示。鉴于使用小鸡内耳外植体的便利性和可及性，我们可以通过证明筛选使用培养基来鉴定毛细胞再生所必需的新分子。

Research

JoVE Journal

Developmental Biology

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

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

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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 ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo Electroporation in the Chicken Auditory Brainstem

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 ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Creating Avian Forebrain Chimeras to Assess Facial Development

创建鸟类前脑嵌合体以评估面部发育

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

在 奥沃 商业肉鸡蛋的喂养：一种影响肌肉发育和生长的准确且可重复的方法

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during ...

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

在蛋和卵外研究中研究鸟类内耳发育的方法