这项工作是由美国国立卫生研究院和PKD的基础ZS支持。所有的动物实验，根据耶鲁动物资源中心（YARC）和机构动物护理和使用委员会（IACUC）的指导方针进行。

第1部分：微量制备，显微注射室板<ol><li>编造加热和拉​​在微量拉拔设备的高硼硅玻璃毛细管（世界精密仪器公司，1B100 - 4）（萨特仪器公司，火焰山/布朗的P - 97）的微量。在培养皿中少量的粘土或胶带上的商店。 </li><li> 1X E3媒体与楔形槽，将成为一个简单的方法（如“斑马鱼书”中描述的）在注射胚胎成型板，倒在1.5％琼脂糖（美国Bioanlytical公司，AB00972 - 00500）让琼脂室板之前，使用和储存在4℃固化。 </li></ol>第2部分：RNA的制备基因功能研究的一个强大的方法是microinject在体外转录的斑马鱼胚胎的上限RNA。有回报上限的RNA的行为同样由于存在第模拟体内发现的真核细胞的mRNA。斑马鱼的研究人员经常利用这种方法，他们感兴趣的基因过表达或misexpress。在这个演示中，我们将microinject EGFP标记的成绩单和使用作为一个成功的注射可见读数的活全胚胎GFP的表达。 <ol><li> 执行体外转录反应第RNA对您感兴趣的成绩单。在我们的实验室中，我们经常插入一个pCS2 +矢量利益的cDNA，并执行在体外大量使用mMessage mMachine SP6套件（Ambion公司，股份有限公司，AM1340）的上限核糖核酸的合成反应。 </li><li>纯化的RNA样品通过的RNeasy试剂​​盒（QIAGEN公司，74104）运行它，或由苯酚：氯仿抽提，异丙醇沉淀。 </li><li>仔细确定，直到准备使用于-80 º C浓度的RNA制备和存储。 </li><li>注射当天，解冻的RNA样品，涡掉以轻心，并简要降速。 </li><li>准备工作液与无菌水，终浓度为0.025 - 0.050％酚红（Sigma - Aldrich公司有限公司，P0290）。酚红作为一个可见的溶液注射到胚胎标记。在这篇文章中，我们将准备与0.050％酚红的100毫微克/ UL EGFP的表达的工作解决方案。 </li><li>冰和返回RNA的股票保持至-80 ° C的工作示例</li><li>继续执行第4部分准备就绪后，注入样品。 </li></ol>第3部分：吗啉的制备吗啉反义寡核苷酸被广泛用于修改有针对性的蛋白质通过阻断翻译或修改前体mRNA剪接2,3基因的表达。在斑马鱼Morpholinos撞倒基因的功能，作为一个强大的反向遗传学工具。在这篇文章中，我们将microinject一个吗啉代寡核苷酸有针对性地PKD2翻译起始位点（5&#39; - AGGACGAACGCGACTGGAGCTCATC - 3&#39;）。基于先前建立的工作，我们预计注入的胚胎表型模仿 PKD2突变的鱼4。 <ol><li>我们经常为了一个吗啉代寡核苷酸300 nmol专门设计一个基因从基因工具的兴趣，LLC（Philomath，或）。 </li><li>添加100微升无菌水，使一个3毫米的原液。分装的解决方案，并在-20 ° C储存，直到准备使用。 </li><li>注射当天，热啉的解决方案在65 ° C为5分钟。立即对齐冰酷和自旋简要。这一步变性任何寡核苷酸二级结构，并确保该解决方案是完全溶解。 </li><li>准备稀释在无菌水和终浓度为0.025啉解决方案的一个可行的解决方案 - 0.050％酚红。在这个演示中，我们将准备与0.050％酚红的0.50毫米PKD2啉的工作解决方案。 </li><li>保持在室温下工作的解决方案。 </li><li>继续执行第4部分准备注入样品时.---- </li></ol>第4部分：与你的工作解决方案灌装的微量<ol><li>切一个与微管钳的远端提示或外科razorblade创建一个开放，这只是在50X放大倍率（尼康仪器公司，SM2645）在解剖显微镜下可见。 </li><li>放置2 UL你的工作解决方案，下降到盖玻片。 </li><li>将微量的背面装有皮下注射针管和轻微淹没远端尖端的微到您的工作液下降至5 mL注射器。小心不要打破的微尖。 </li><li>拉动注射器的柱塞，通过末端的微虹吸式的工作方案。 </li></ol>第5部分：校准微量注射量<ol><li>将下降一个千分尺幻灯片矿物油（美国生物分析公司，AB00921 - 00025）（FISHER，12 - 561 - SM1）。 </li><li>打开电源和空气供应的压力脉冲的微量进样装置（世界精密仪器公司，PV830）。 </li><li>将微量微量微量进样装置的持有人。微量进样器和放电按脚踏激活监管的压力。 </li><li>解剖显微镜下，测试使用微量进样器放置到你的工作解决方案微米油下降的注射量。测量液滴的直径，因为它作为一个石油顶部的球体浮。 </li><li>调整的时间和仔细校准你的注射量，注射压力。这一步艾滋病的显微注射实验的可重复性。在这个演示中，所有microinjections会做一个直径0.15毫米（约1.76 NL量）下降。 </li><li>一旦你的注射量已经校准，使用微量进样器上的“保留”旋钮，以防止回填和泄漏的微。 </li></ol>第6部分：准备受精卵显微注射的斑马鱼胚胎<ol><li>斑马鱼将在每天早上的头几个小时随机交配。收集1 - 细胞阶段的胚胎和它们放置在1X E3的介质。 </li><li>使用3毫升转移的吸管（碧迪医学实验，357524），安排沿显微注射室板的楔形槽胚胎。 </li><li>取出介质，使胚胎浅淹没，不淹。这一步，​​艾滋病，以及由微绒毛的渗透，解决胚胎槽的底部。 </li></ol>第7部分：通过蛋壳显微注射<ol><li>操作与微管，使1 - 细胞期胚胎的细胞质解剖显微镜下可见的胚胎。小心不要打破的微尖。 </li><li>渗透与微绒毛膜，然后蛋黄，以注入胚胎。在这个演示中，我们将首次直接进入下面的单元格蛋黄显微注射，使细胞质的流动和扩散，使进入细胞的工作液。这种流动是可见的，由于加入到工作液的酚红。 </li><li>我们也将展示直接注射到细胞的细胞质。直接进入细胞内显微注射的是更强大，但耗时适当的胚胎的方向和显微注射技术是必需的。由于细胞膜比蛋黄膜更严格，它往往是快速进入蛋黄的微量达到细胞质。对槽的底部定向动物极和稳定手中的工作，可能有助于在这种注射方法。 </li><li>显微注射后，放入一个培养皿的胚胎带有E3介质，并培育他们在28.5 ° C的正常发展。 </li><li>在接下来的几个小时，天的胚胎发育观察您感兴趣的表型的胚胎。 </li></ol>第8部分：代表结果 EGFP的mRNA表达：要验证显微注射的成功，我们会密切监察在盾构阶段（〜6 HPF） 开始，在体内的全胚胎荧光显微镜的发展胚胎中的表达绿色荧光蛋白（莱卡微系统公司， MZFLIII）。构建的表达在胚胎发育的早期事件，是最强大的的，往往会在发展过程中减少注入上限RNA逐渐退化，取决于所表达的蛋白质的稳定性。 PKD2翻译阻断啉：根据先前公布的结果，我们预计PKD2 morphants模仿背侧体轴曲率PKD2突变的斑马鱼和目前的肾囊肿，如发现在大约2 - 3 DPF 4。 <img src="/files/ftp_upload/1113/1113fig1.png" alt="图1" /> 图1：注射EGFP的mRNA和PKD2八月啉代表性的结果。 （一）制表胚胎显微注射的1 -细胞阶段与0.17 EGFP的表达吴，并在1 DPF可视化在体内GFP的表达。 （B，C）制表胚胎显微注射的1 -细胞期在0.50毫米〜1.7 NL一个PKD2翻译阻塞啉和背身体曲率拿下4 DPF。

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	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Nasevicius, A., Ekker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat Genet. 26, 216-220 (2000).</li><li>Draper, B., Morcos, P., Kimmel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+zebrafish+fgf8+pre-mRNA+splicing+with+morpholino+oligos:+a+quantifiable+method+for+gene+knockdown.">Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown.</a> Genesis. 30, 154-156 (2001).</li><li>Sun, Z., 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=A+genetic+screen+in+zebrafish+identifies+cilia+genes+as+a+principal+cause+of+cystic+kidney.">A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney.</a> Development. 131, 4085-4093 (2004).</li><li>Stuart, G., McMurray, J., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication,+integration+and+stable+germ-line+transmission+of+foreign+sequences+injected+into+early+zebrafish+embryos.">Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.</a> Development. 103, 403-412 (1988).</li><li>Stuart, G., Vielkind, J., McMurray, J., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+lines+of+transgenic+zebrafish+exhibit+reproducible+patterns+of+transgene+expression.">Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.</a> Development. 109, 577-584 (1990).</li><li>Culp, P., N&#252;sslein-Volhard, C., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+germ-line+transmission+of+plasmid+DNA+sequences+injected+into+fertilized+zebrafish+eggs.">High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs.</a> Proc Natl Acad Sci U S A. 88, 7953-7957 (1991).</li><li>Strehlow, D., Heinrich, G., Gilbert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fates+of+the+blastomeres+of+the+16-cell+zebrafish+embryo.">The fates of the blastomeres of the 16-cell zebrafish embryo.</a> Development. 120, 1791-1798 (1994).</li><li>Helde, K., Wilson, E., Cretekos, C., Grunwald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+early+cells+to+the+fate+map+of+the+zebrafish+gastrula.">Contribution of early cells to the fate map of the zebrafish gastrula.</a> Science. 265, 517-520 (1994).</li><li>Lin, S., Long, W., Chen, J., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+germ-line+chimeras+in+zebrafish+by+cell+transplants+from+genetically+pigmented+to+albino+embryos.">Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos.</a> Proc Natl Acad Sci U S A. 89, 4519-4523 (1992).</li><li>Long, Q., 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=GATA-1+expression+pattern+can+be+recapitulated+in+living+transgenic+zebrafish+using+GFP+reporter+gene.">GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene.</a> Development. 124, 4105-4111 (1997).</li><li>Murphey, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+screening+in+the+zebrafish.">Small molecule screening in the zebrafish.</a> Methods. 39, 255-261 (2006).</li></ol>

微量注射到斑马鱼的胚胎是一个探索发展的一个特定基因的作用以及建立和强大的技术。应用范围包括过度表达，错误表达，和您的利益，以及多个基因之间的互作分析的基因敲除实验。微量注射在斑马鱼已被广泛用于产生转基因动物，和映射在 5,6,7,8,9囊胚早期胚胎细胞的命运。此外，这项技术的应用作为一个细胞移植方法 10种系嵌合体的关键一步。 一个替...

在协议中列出的所有其他材料。

microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos.

调查在开发过程中的一个基因的作用的一个必不可少的工具，来进行基因敲除，的过度表达，和错误表达研究的能力。在斑马鱼（<em&gt;斑马鱼</em&gt;）的RNA，DNA，蛋白质，反义寡核苷酸等小分子显微注射到发育中的胚胎的研究人员提供了一个用于探索基因功能的快速和强大的检测<em&gt;在体内</em&gt;。在这个视频，文章，我们将演示如何准备和microinject<em&gt;在体外</em&gt;合成EGFP的表达和翻译阻断吗啉代寡核苷酸对<em&gt; PKD2</em&gt;，与常染色体显性多囊肾病（ADPKD）为1 - 细胞期的斑马鱼胚胎，相关的基因。然后，我们将分析的mRNA和核实绿色荧光蛋白表达和表型分析啉microinjections的成功。这项技术的广泛应用，包括转基因动物和种系嵌合体的产生，细胞命运的映射和基因筛查。在此我们描述了过度表达EGFP和击倒的协议<em&gt; PKD2</em&gt; mRNA和吗啉代寡核苷酸注射。

Watch this Scientific Journal Video about Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos. at JoVE.com

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

显微注射是外来物质引入斑马鱼胚胎受精的完善而有效的方法。在这里，我们展示了一个强大的执行mRNA表达显微注射技术，和吗啉代寡核苷酸在斑马鱼的基因敲除研究。

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 ...

microinjection-mrna-morpholino-antisense-oligonucleotides-zebrafish

Research

JoVE Journal

Biology

36.6K 次观看.  Yale University School of Medicine. 显微注射是外来物质引入斑马鱼胚胎受精的完善而有效的方法。在这里，我们展示了一个强大的执行mRNA表达显微注射技术，和吗啉代寡核苷酸在斑马鱼的基因敲除研究。

视频: Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

埃及斑蚊胚胎显微注射获得转基因蚊子

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

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 ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.