Name: reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva
Uploaded: 2014-06-17T15:00:00
Description: 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

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...

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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。

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			<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 ...

Research

JoVE Journal

Biology

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

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

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

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

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

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

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

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

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

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 ...

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

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 ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...