这项工作是由德意志研究联合会（DFG）的支持下，我们要感谢Ayele Tsedeke Taddese寻求技术支持。

1（图1）的玻璃针的下游<ol><li>用玻璃毛细管，直径0.75毫米以制备注射针头（ 图1A）。 </li><li>将玻璃毛细管进针车夫和拉针使用以下参数：加热循环值：463;拉动周期值：230;速度：150毫秒;时间：150毫秒（ 图1B）。 </li><li>打破与制表师用镊子拉玻璃毛细管，产生下用测微目镜一个立体镜20微米直径的针。 </li><li>使用车床用湿润的橡胶纺车，是加强针，并产生一个20微米的伞（ 图1C和1D）。注意：夏普，斜面针改善易于插入到心室，从而最大限度地减少对组织的损害，并允许在穿孔的肌肉切除针（ 图1E-G）之后重新密封。 </li></ol> 2。Prepar吗啉溶液的振动性<ol><li>通过溶解在1×磷酸盐缓冲盐水（PBS）中的冻干的吗啉代，以7.5毫米的最终浓度制备的吗啉代库存。 [参见制造商的说明资料（材料表）]。 </li><li>通过混合2.5微升啉原液（7.5毫摩尔）与2.8微升的Endo-Porter的储备溶液（1毫摩尔）（见材料表）为3.5毫啉和0.5mM的Endo-Porter的终浓度制备的吗啉注射溶液。 </li></ol> 3，吗啉注射液的制备（图2） <ol><li>加载斜角玻璃针与5微升该溶液的使用微量用10微升微加载枪头。 </li><li>插入玻璃针插入连接于气动微微泵（ 图2A-C）的微操作的持针器。 </li><li>将针保持器旁边的显微镜，使针只需要在一个平面方向上移动，以将其插入幼虫（ 图2A）的心室。 </li><li>调整角度为注射剂在45°左右。 </li><li>将微量值如下：保压压力：20磅每平方英寸（psi）;喷射压力：15磅;门的100毫秒范围，1.9周期值（对应于10.9毫秒）。 </li><li>使用标准的微波，产生20ml的1.5％（重量/体积）的凝胶在1×PBS中熔化琼脂糖。倒入熔化的凝胶放入一个10cm培养皿。放置槽注入的形式进入温暖的琼脂糖这样一旦硬化，琼脂糖将有沟，其中镇静幼虫将放在11（ 图2D和2E）。 </li></ol> 4，注射剂（图3） <ol><li>麻醉幼虫在100毫升水族箱的水与20 mg / L的三卡因（1 - 乙基-M-氨基苯甲酸酯甲磺酸酯），直到他们停止响应触摸。 </li><li>用塑料巴斯德pipett以e为镇静幼虫小心转移到湿琼脂糖模具的凹槽，以使腹侧面朝靠在凹槽（ 图3A）的垂直琼脂糖壁。 </li><li>将琼脂糖板的立体显微镜下，使心室背向注射针（ 图3A）。 </li><li>降低针以将其插入到心脏心室。将针头只有1-2微米入脑室，注意不要插入针太深（ 图3B和3C）。 </li><li>一旦将针插入时，吗啉溶液与4-6个脉冲，每个提供3 NL溶液注入脑室，与等待时间间隔，以允许心脏（ 图3D和3E）的清除。 </li><li>注射后，取出针（平行插入的平面），然后小心地用塑料巴斯德吸管充满E3媒体BAC转幼虫K表到含有新鲜的E3媒体的培养皿。 </li><li>将针头插入含有1x PBS的培养皿中，以防止针的干燥输送所注入的幼虫时。 </li><li>重复步骤4.1-4.6每12-24小时进行的实验的持续时间。注意：重​​复注射，确保在细胞中的吗啉代基的吸收和维修。 </li></ol> 5，注射的分析（图4） <ol><li>在100毫升的水族箱的水与20 mg / L的三卡因麻醉的鱼，直到他们停止响应触摸。 </li><li>将幼虫横向放置在平坦的湿的1.5％（重量/体积）的琼脂糖板覆盖E3介质。 </li><li>使用带有明和荧光成像立体显微镜图像幼虫。注：由于幼虫是透明的，荧光素标记的吗啉代应该是荧光绿色的血管遍布直接注射后动物可见。此荧光将进一步分散到血管组织中15分钟（ 图4A-4C）。 </li></ol>再生向外长出6。评估<ol><li>使用斐济图片Ĵ免费软件（http://fiji.sc/Fiji）评估拍摄的图像。为了研究再生，使用线追踪工具来确定再生的增长发生在控制和吗啉注射幼虫的数量。 </li><li>要校正的图像，打开通过拖放文件到主窗口斐济在斐济的原始图像文件之一。 </li><li>要设置刻度以下所有图片，请在主菜单中的“分析”。 </li><li>在下拉菜单中点击“设置比例”。 </li><li>在这个窗口中，打开通过单击复选框“环球”选项。 </li><li>现在打开通过拖放（参见步骤6.1）再次测量再生生长量的图像。 </li><li>请在工具栏上的“写意选择”工具。 </li><li>包围要测量的区域（ 连接gures 4J-4L）。 </li><li>按Ctrl + M。这将打开一个新的“结果”窗口显示在正方形像素的包围区域的价值。 </li></ol>

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作者什么都没有透露。

脑室注射提供了一个快速和可靠的评估方法，用于测试基因的功能在以后的身体内环境稳定的发展阶段，或在不影响胚胎发育过程中的基因功能。为了确保这项技术的成功，人们应该知道的四个关键点：1）针头大小，2）晒出针，3）尽量减少体积，并在镇静解4）最小的曝光时间。针太小会经常堵塞，而针是太大，会损伤脑室，引起出血过多。而20微米的建议，针不应该超过五分之一的心室腔的大?...

器官和附肢再生的生存和健康极其重要;然而，一些脊椎动物包括人类有限的再生能力。虽然一些动物模型中存在有大量的再生能力，反向遗传技术的器官和附肢再生过程中，评估基因的功能仍十分有限或不存在。因此，需要采取新方式来剖析再生的分子生物学在这些模式生物。 斑马鱼是一种行之有效的模式对于了解细胞和器官和附肢再生的，不仅是因为再生多个器官，组织和附属物其显著的能力，而且也为1，分子生物学，因为几个转基因鱼系的存在是为了追踪细胞和以过表达基因构建体2，3。然而，在斑马鱼幼鱼基因抑制主要限于overexpre裂变的显性负性结构，这并不适用于所有感兴趣的基因或转基因，其产物可以获取功能获得的效果是不反映基因的内源活性。因此，另一种方法，通过基因敲除或敲除专门去除基因的表达是需要克服这些问题。 基因特异性使用TALENS存在作为反向遗传学手段敲除基因的功能定位;然而，这淘汰赛策略是非常频繁局限于功能评估在早期胚胎发育，因为基因的初步要求预防胚胎发育的进一步发展。因此，学习后的现象，如使用TALENS发展后再生或器官的动态平衡被排除4，5。因此，另一种基因去除的策略是必要的，早期的发展目标后，基因的功能，以评估完全形成器官和结构基因的要求。 </P&gt; 吗啉注射已被证明是有效的靶向基因在几个成年器官和成年再生鳍6-8，但是这些方法都需要电穿孔和许多内部器官都难以电穿孔或者由于它们的位置或由于其电气灵敏度中断。此外，在一些幼虫组织是难以直接注入，由于直接喷射可能会破坏其结构的完整性，或者因为它们的大小被限制。幼虫的尾鳍就是这样的一个结构，因为直接注射到finfold是不可能的。因此，另一种电穿孔和直接注射是需要的靶基因在组织中，要么太小注入或不能被电穿孔。 为了幼虫尾鳍的再生过程中对目标和抑制特定基因的功能，我们已经修改现有啉技术允许一个基因功能的过程中下旬上演幼虫尾鳍再生ssessment。这种方法采用荧光素标记的吗啉脑室交付9用Endo-Porter的转染试剂10在一起。一旦进入心室，吗啉代的Endo-波特混合物迅速传遍整个幼虫通过脉管系统，并进入到先前已不可能对靶组织。这种注射方法可以被修改以靶基因在特定组织和很可能可以在目前缺乏的反向遗传学方法来抑制基因的功能的其他动物模型中被应用。因此，它提供了一个快速简便的方法与范围广泛的使用过程中在幼虫期一般器官平衡和再生，立即研究基因功能的潜力。

<table border="0" cellpadding="0" cellspacing="0" style="width:672px;" width="673">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagent/Material</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
			<td style="width:171px;">Applichem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For anesthesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Petri Dishes</td>
			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
	</tbody>
</table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

斑马鱼是了解细胞和器官和附肢再生的分子生物学研究的重要模式。然而，分子的策略采用反向遗传学还没有得到充分的研究，以评估在斑马鱼胚胎发生后幼虫阶段再生或组织稳态的基因功能，而斑马鱼幼体中的多个组织是困难的目标。的基因特异性吗啉脑室注射对当前不能在这些阶段中，以基因组靶基因斑马鱼在时间上控制的方式提供另一种方法。此方法允许完全分散和吗啉代的后续掺入到整个身体的各种组织，包括在以前是不可能达到如那些在幼虫尾鳍，经常用于非侵入性地研究的组织再生的结构的结构。在幼虫finfold再生激活一些基因也PRESEN吨再生成年脊椎动物组织中，所以幼虫是一个有用的模型，以了解再生的成年人。这啉分散法允许快速和容易识别所需的幼虫组织的再生，以及其他生理现象后，胚胎发育调控组织稳态的基因。因此，该发送方法提供了一种目前所需要的策略临时控制到胚胎后，基因功能的评价。

锋利，斜面注射针很容易放置在斑马鱼幼虫的心室背侧接触（ 图3A）时 ​​。的心脏继续泵送和血流维持尽管针（ 图3B和3C）的存在。小心打针不扰乱心室或心脏收缩（ 图3D），尽管注射吗啉代入心脏（ 图3E）的形态。 在几分钟内，吗啉代的Endo-波特溶液通过脉管系统（ 图4A和4B）分布?...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

一个适应性强的反向遗传学方法，斑马鱼在后来的发展和生理平衡，如使用基因特异性吗啉脑室注射组织再生的阶段来评估基因的功能。

反向遗传吗啉使用方法心脏心室射出转染多难靶组织中的斑马鱼幼虫

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

10.5K 次观看.  Technische Universität Dresden. 一个适应性强的反向遗传学方法，斑马鱼在后来的发展和生理平衡，如使用基因特异性吗啉脑室注射组织再生的阶段来评估基因的功能。 

视频: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...