Name: analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle
Uploaded: 2013-03-31T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

我们感谢菲奥娜优良的实验室支持和斑马鱼的保健福利。这项工作是一个AHA博士前奖学金GW（11PRE5730027）的和NHLBI补助金的HJY（R01HL66292）和JDA（R01HL095690）。

概述特定阶段的斑马鱼胚胎注射液反义吗啉代寡核苷酸（MO），结合到靶mRNA和蛋白表达，转录干扰，被广泛用于基因敲除（损失函数）的研究斑马鱼13,14。基因的工具，LLC提供的MO，无论是羧基发出绿色荧光的或丽丝胺（发出红色荧光）的检测MO注射的胚胎中，使用荧光显微镜的标签。通过注入MO到蛋黄细胞在斑马鱼发育的不同阶段中，它能够提供的MO胚胎的具体的室中（...

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使用特定阶段的目标MO注射到DFC / KV细胞系是一个非常有用的方法来研究基因功能的细胞自主性，避免多效性所造成的全球基因敲除的表型。然而，这些注射可以在技术上具有挑战性。注射液的MO之间的256细胞和1000细胞的阶段，可能会导致在三种可能的结果：1）在MO仍然聚集在注射部位，2）的MO整个蛋黄扩散并进入DFC / KV细胞或3）的MO整个蛋黄，进入DFC / KV细胞和胚胎干细胞的扩散。因此，在这个?...

<table border="1"><tbody><tr><td>的试剂/材料名称</td><td>公司</td><td>目录编号</td></tr><tr><td>标准控制寡里沙明标记</td><td>基因工具，LLC </td><td></td></tr><tr><td>定制Rock2b吗啉代寡核苷酸</td><td>基因工具，LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0.5微米的微球</td><td> Polysciences的公司</td><td> 24054 </td></tr></tbody></table>

analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle

内部器官，如心脏，大脑和肠道左右（LR）为他们的正常功能是至关重要的不对称性。运动纤毛参与建立LR的不对称性在脊椎动物胚胎，包括老鼠，青蛙和斑马鱼2-6。这些&#39;LR的纤毛&#39;产生不对称的流体流动是必要的，以触发一个保守的不对称交点（TGF-β超家族）的信号级联在左侧侧板中胚层，这被认为是提供开发机关7 LR构图信息。因此，理解机制LR构图，它是必不可少的，以确定基因调节的LR纤毛细胞的组织，运动和长度LR纤毛和他们的能力，以产生强大的非对称流。 在斑马鱼胚胎中，LR纤毛位于巨噬泡（KV）2,4,5。 KV是由一个单一的层monociliated上皮细胞，附上一个充满液体的腔。 KV是来自一组被称为的背先行者细胞（DFCS）迁移外包阶段在背胚率在8,9〜20-30细胞的命运映射。在早期的体节阶段，DFC的集群，并分化成纤毛上皮细胞，形成KV中的胚胎10,11 tailbud。结合的光学透明度和快速发展的斑马鱼胚胎斑马鱼KV一个很好的模型系统来研究，LR纤毛细胞能够识别和跟踪八间指定流感诊所求诊。 有趣的是，DFC / KV细胞谱系的祖细胞保留蛋黄细胞的细胞质之间的桥梁，受精后4小时（HPF）的，而蛋黄细胞和其他接近8高倍视野2后的胚胎干细胞的细胞质之间的桥梁。利用这些细胞的桥梁，我们开发了一个特定阶段的喷油策略提供吗啉代寡otides（密苏里州）专门DFC的和击倒的目标基因的功能，在这些细胞中12。这技术创造嵌合体胚胎基因的功能被撞倒在发展的背景下，野生型胚胎的DFC / KV血统的。在KV，我们要分析非对称流体流动注射荧光微球到KV珠流明，并记录运动时，通过电视显微镜2。流体流动是易于可视化和可量化的跟踪珠位移随着时间的推移。 在这里，使用特定的阶段，的DFC-针对性的基因敲除技术和注射荧光微球KV可视化流程，我们提出了一个协议，提供了一个有效的方法来描述一个特定基因的作用在KV的发育和功能。

图1给出了一个流程图，用于测试基因功能DFC / KV细胞和如何引入荧光珠的可视化流体流的喷射策略阶段特异性的MO注射提供了一种有用的方法来分析基因功能的特定的室中的胚胎。在KV。在成功阶段特异性注射胚胎的是，荧光的MO分布示意性地示出在图2中，在图1D-F和在活胚胎。一个不成功的MO注射，在其中的MO仍然在蛋黄细胞聚集， 如图2D</...

Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

纤毛产生的流体流动在枯否囊泡（KV）控制的斑马鱼胚胎的左右模式。在这里，我们描述了一种技术，调节基因的功能，特别是在KV细胞。此外，我们展示了如何提供KV可视化流体流动的荧光珠。

基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Research

JoVE Journal

Biology

Microinjection of Zebrafish Embryos to Analyze Gene Function

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

科研

生物学

Induction and Testing of Hypoxia in Cell Culture

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

联合基因组学，代谢组学和信息通过植物基因功能的注解

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ Hybridization for Sipunculus nudus Coelomic Fluid

西普库鲁斯核素体液的原位杂交

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...