Name: in ovo intravascular injection in chicken embryos
Uploaded: 2022-06-03T13:00:00
Description: Watch this Scientific Journal Video about In Ovo Intravascular Injection in Chicken Embryos at JoVE.com

这项工作得到了国家自然科学基金（31972547）的支持。我们感谢王静的文案编辑和美国华盛顿州立大学马利克·唐利克的画外音。

所有涉及动物护理和使用的程序均符合美国国立卫生研究院指南（NIH Pub. No. 85-23，1996年修订）和鸡胚胎方案均获得中国扬州大学实验动物管理和实验动物伦理委员会的批准（No.201803124）。
1. 受精卵收集和制备
注意：与哺乳动物不同，鸡在单个卵巢中有数百万个卵泡，但只有少数这些卵泡成熟到足以排卵。每个卵泡含有一个卵母细胞或生殖细?...

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在 卵 子血管内注射鸡胚胎的方法针对要转移到胚胎中的外源性物质（载体，病毒或PGC）进行了优化。基于该方法，我们构建了具有稳定基因过表达或干扰的鸡胚模型（SpinZ，JUN，UBE2I等）。17，18，19.这些成熟的模型证明了这种方法的可行性。此外，我们不仅将分离的PGCs转移到受体的胚胎性腺上，而且还成功地用诱导的P...

几个世纪以来，鸡胚已被广泛用于发育，免疫，病理和其他生物学应用1，2，3。它们在毒理学和细胞生物学研究中比其他动物模型具有许多固有的优势4.鸡胚易于获取，可以在 体外 操纵并在任何发育阶段直接观察，这提供了一个方便的胚胎研究模型系统。
一般来说，目前的鸡胚胎递送方法，如电转染和胚芽下腔注射，具有局限性，例如需要专业设备和设计的程序，并且由于蛋黄和蛋白5，6，7的存在而效率低下。在这里，我们展示了一种将外源性物质输送到鸡胚中的简单有效的处理方法。这可以成为用于发育生物学研究的强大工具。注射的材料 通过 血液循环扩散到整个胚胎。在鸡胚胎的早期发育过程中，PGCs可以通过血液迁移，定植于生殖器脊，然后发育成配子，这为传递外源性物质提供了有价值的可能途径8。现在，该方法已广泛应用于研究基因功能、胚胎和发育生物学，以及嵌合和转基因鸡的生产9，10，11。
在蛋黄 血管内注射鸡胚中是一种完善和常用的方法12，13，14。在本文中，我们展示了该方案的全面描述，包括注射材料，部位，剂量和代表性结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescence macro-microscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Narishige</td>
			<td>G1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>12566014</td>
			<td>liposome</td>
		</tr>
		<tr>
			<td>pEGFP-N1 vector</td>
			<td>Clontech</td>
			<td>#6085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit&#160;</td>
			<td>Sigma</td>
			<td>PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>pLVX-EGFP lentivirus vector</td>
			<td>Addgene</td>
			<td>128652</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic Microinjector</td>
			<td>Narishige</td>
			<td>IM-11-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

in ovo intravascular injection in chicken embryos

作为胚胎生物学的经典模型系统，鸡胚已被用于研究胚胎发育和分化。将外源物质递送到鸡胚中，对于研究胚胎发育过程中的基因功能、转基因育种和嵌合体制备具有很大的优势。在这里，我们展示了 在卵子 血管内注射中的方法，其中外源性物质如质粒载体或修饰的原始生殖细胞（PGCs）可以在早期发育阶段转移到供体鸡胚胎中。结果表明，通过背主动脉和头部的血管内注射允许注射的物质通过血液循环系统扩散到整个胚胎中。在所提出的方案中，通过观察胚胎中的荧光，确定了外源质粒和慢病毒载体引入的疗效，以及注射的外源PGC在受体性腺中的定植。本文介绍了该方法的详细程序，从而为研究基因功能，胚胎和发育生物学以及性腺嵌合鸡的生产提供了极好的方法。综上所述，本文将使研究人员 能够在卵 子血管内将外源物质注射到鸡胚中，取得巨大成功和可重复性。

我们在这里展示了在 卵子 血管内注射鸡胚。血管内注射的过程示意图如图 1所示;在我们的研究中，我们使用各种外源性溶液来测试和验证注射。
为了更好地可视化注射的材料，台盼蓝（0.4%）作为示踪剂注射到胚胎中。观察到示踪剂（蓝色）通过背主动脉或头部注射 通过 血液循环扩散到整个胚胎（图2）。在两个不?...

Watch this Scientific Journal Video about In Ovo Intravascular Injection in Chicken Embryos at JoVE.com

In Ovo Intravascular Injection in Chicken Embryos

本文的总体目标是描述如何在卵细胞内将外源性物质注射到鸡胚 中 。这种方法对于研究鸡胚的发育生物学非常有用。

在 奥沃 鸡胚血管内注射

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

您好，我是扬州大学的Kai Jin。我将代表我们的研究小组介绍一种强大的鸡胚在发育研究中的工具。鸡胚是一种经典的发育模型，一直被用来研究发育和分化，事实上，实验室对鸡胚的无关措施是发育生物学的宝贵工具，如基因转染、转基因育种、性腺透壁制备等。在我们的实验室中，我们已经建立了μ型注射方法，但对于许多研究人员来说，注射的细节不清楚，很难排队。因此，在本视频中，我们将展示在蛋黄鸡胚发育的早期阶段血管注射的细节。如果您对此感兴趣，请随时与我们联系。在这里，我们展示了协议的细节。受精卵收集和制备。首先，与哺乳动物不同，鸡在单个卵巢中有数百万个卵泡，但只有少数这些卵泡成熟到足以排卵。每个卵泡含有一个卵母细胞或生殖细胞。一旦卵泡成熟并释放其卵黄，它就会被纳入输卵管的漏斗中。当卵泡进入输卵管的颈腹部时，精液与母鸡体内的鸡蛋结合，母鸡体内的钙形成一个壳，包裹受精卵，形成体内软壳蛋。钙壳逐渐变稠，直到产生卵子。收集受精卵，学术上称为胚胎卵，并将它们整齐地放在37.8摄氏度和60%湿度的孵化器中的架子上。胚胎在合适的条件下孵育。经过两天的孵化，出现了称为原始心脏的小脉动红点。这个过程被称为樱桃打。在大约2.5天时，血管可见，心脏和身体部分已经形成。针和质粒制备。我们使用玻璃毛细管作为针头。注射前准备外径为1毫米，内径为0.6毫米的玻璃毛细管。外源性物质通常是质粒、包装慢病毒和原始生殖细胞或PGCs。在本视频中，我们使用台盼蓝来展示注射材料的痕迹。窗口。在暴露胚胎之前，我们需要对胚胎进行消毒，以防止细菌污染。这是通过在卵子开口处用75%酒精棉球轻轻擦拭胚胎表面来完成的。消毒后，用镊子轻轻敲击蛋壳的钝端，并用镊子小心地在蛋壳表面夹一个0.5厘米×0.5厘米的孔，露出胚胎。卵内血管内注射。在显微镜下观察视图，调整显微镜的焦点，首先定位胚胎，然后找到准备注射的鸡胚胎的血管。将带有外源物质的针头对准血管，注意针头与注射的血管平行。将针头轻轻插入血管，然后打开空气泵。此时，异物进入动脉并随着心脏的跳动，通过动脉循环回胚胎，然后循环到静脉。有两种类型的血管注射。一种是头部注射，其中异物从胚胎头部的静脉注射并流回心脏。另一种是背主动脉注射，其中异物随着心脏的跳动而循环到胚胎。随着胚胎心脏的跳动和血液的循环，我们可以清楚地看到，异物被动脉末端的毛细血管接收，进入静脉，然后从静脉返回胚胎，表明注射的异物有效地输送到胚胎的每个部分。注射后，从立体显微镜中取出卵子，用小剪刀剪下一块三厘米长的医用胶带，然后将胶带贴在开口处。用剪刀轻轻刮拭胶带以挤出任何气泡，然后切开另一块胶带以将开口密封成十字架。密封开口后，将注射的胚胎小心地放回培养箱中。在这里，我们展示了注射后鸡胚的代表性图像。第一张图像显示了慢病毒质粒注射的结果。立体荧光显微镜下的绿色荧光表明注射的质粒在胚胎中成功表达。这表明血管内将质粒注射到鸡胚中的可行性。第二张图显示了PGC注射的结果。PGC被标记为RFP。在这张图片中，我们可以看到注射的PGC，它们是这里的红点，位于受体的性腺中。这表明供体PGC能够有效地进入受体的性腺并定植。总之，该协议介绍了卵内血管内外源物质，质粒或PGCs注射到鸡胚中的过程。这里展示的方法易于遵循和学习，值得在许多领域应用。这就是我想与大家分享的全部内容。我希望这种方法能够促进您作为设置鸡胚和发育生物学的强大研究工具的工作。感谢您观看此视频，祝您研究顺利。

Research

JoVE Journal

Developmental Biology

In Ovo Intravascular Injection in Chicken Embryos

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 ...

科研

发育生物学

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

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 ...

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 ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

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 ...

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 ...

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

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

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 ...

在 奥沃 鸡胚血管内注射

在奥沃鸡胚血管内注射