我们承认他们的技术援助马布利实验室和Narie斯托勒成员，和美国心脏协会（NCRP的科学家开发格兰特0635363N）和国家心脏，肺和血液研究所（批准SCCOR射频消融HL02 - 027）提供资金。

第1部分：蛋的生产和收集<ol><li>注射前的晚上，设立鱼繁殖与到位的分隔坦克。为了增加总蛋生产，鱼可以成立一个男性两名女性的比例如果需要。 </li><li>第二天早晨，打开后，房间的灯，拉几个坦克的分隔，并允许不受干扰的交配时间约20分钟。 </li><li>使用一个过滤器，收集从养殖笼具，蛋和蛋水冲洗。鸡蛋与蛋水倒入培养皿中，并清除未受精卵，并用移液管的碎片。鱼可以集结更大的坦克，以产生额外轮注射鸡蛋。鸡蛋收集的时间调整，以便最大的鸡蛋生产的数字，而让他们通过单细胞阶段。 </li><li>放置在一个100mm的培养皿倒盖的显微镜幻灯片。使用移液管排队反对方形成一个单一的列的幻灯片的鸡蛋。按Kimwipe对一侧对面的鸡蛋，从幻灯片中删除多余的蛋水。 （图1A） </li></ol>第2部分：针拉，装载和准备<ol><li>随着微管拉马，拉进两针，并存储在150毫米培养皿一个1.0毫米OD玻璃毛细管，通过铺设橡皮泥斜坡。针可以提前拉。 </li><li>回程针与3μL注射使用microloader吸管材料。直到有很少或根本没有泡沫，其余摇向针尖丸。 </li><li>打开气源和微量。针插入到微量，并确保一个密封的房屋内。检查的显微操纵器是在一个适当的位置，让一个广泛的运动和调整。针尖带入飞机的显微镜，高走下舞台，和上最薄的提示地区为重点。用一双锐利的镊子捏针在一个点上，叶针窄到足以刺穿蛋壳和蛋黄，但仍然能够提供一个一致的珠子大小的。可用于计算每次注射量的矿物油微米的下降。当注射入油，珠直径0.1毫米的有500注射材料的PL（图1B）;通常用于500 PL或1nL注射量。踩下脚踏板和监察珠的大小，而削减针和调整所需的注射压力。理想的注射量，将填补蛋量的约10％。针尖的质量是至关重要的，以缓解注射液的质量和结果的一致性。 </li></ol>第3部分：注射液<ol><li>确保已不是过去的四细胞阶段的胚胎。理想的情况下，应在单细胞阶段的胚胎。 </li><li>对鸡蛋列下针，在你的对面手的菜。 </li><li>皮尔斯绒毛膜表面的任何破碎或撕裂卵黄囊看，进入一个平稳中风蛋黄。注入注料入蛋黄（图1C）。避免注入气泡或伸展蛋黄作为可致死胚胎。工作的路线，调整压力需要保持一致的珠子大小，使用枪头，消除看受精，或在注射过程中破坏的鸡蛋。 </li><li>鸡蛋列完成后，用一个鸡蛋水的温柔流移动到一个干净的培养皿中注入鸡蛋。必要时重复。保持几个uninjected胚胎作为对照。在第一天结束时，去除死胚胎，并记录注射胚胎的数量。定期更换菜蛋水，以减少感染的机会。 </li></ol>第4部分：代表结果根据胚胎被注入的是什么，可能生存在一个比他们uninjected的兄弟姐妹率较低。幸运的是，它很容易注入大量的，所以这是很少见的问题。这是正常的morphants表现出稍微推迟发展。 为了说明显微注射的结果，我们使用此协议来操作的蛋白质的玻璃心（HEG）水平。 HEG是一种跨膜蛋白，心脏的正常发展所需的。在其缺席的情况下，胚胎发育与巨头的流入大片和商会1心。我们对heg_e3i3_egfr1和heg_e4i4_egfr2，HEG，注入浓度在500微米的两个以前未描述的morpholinos。在受精后2天，出现uninjected兄弟控制正常，如预期（图2A和B）。 heg_e3i3_egfr1注入胚胎脑水肿（图2c）。虽然大多数这些morphants心中已经改变了形态，他们的商会是正常大小的（图2d）。胚胎注入heg_e4i4_egfr2展览变量的心脏缺陷。一些看似正常的，而其他的大量流入大片和适度扩大的心房（图2E和F）。 HEG突变，正如先前报道1，有一个巨大的扩大的流入道和心房（图2G和H） 。 我们也注入400纳克/μLHEG全长的cDNA转录的mRNA与野生型胚胎。而uninjected控制正常发展（图3A和B），胚胎注入HEG的mRNA表现出频谱范围从野生型的外观，严重的独眼畸形的表型，缩短主体轴，头部和身体坏死，和中线缺陷（图3C和D）。 <img src="/files/ftp_upload/1115/Figure%201%20FINAL.png" alt="" /> 图1。注入胚胎在培养皿显微镜幻灯片（一）针对一字排开。注射量是由放在微米矿物油注射。注射量500 PL，这是常用的，有一个直径为0.1毫米（二）。注射后，立即啉或mRNA作为一个蛋黄（三）圈点当场可见。 <img src="/files/ftp_upload/1115/Figure%202%20FINAL.png" alt="" /> 图2。uninjected胚胎在受精后2天，没有严重的缺陷（A）或心脏表型（二）。与吗啉heg_e3i3_egfr1注射的胚胎脑水肿（箭头），但通常大小的心腔（四）。与吗啉heg_e4i4_egfr2注入了一些胚胎扩大流入大片和心房（E，F）。 HEG突变体的严重扩大的流入大片和心房（G，H）。 （一），（三），（E）和（G）4X DIC的图像;（二），（四），（F）和（H）20X DIC的图像。 A =心房，=流入道。 <img src="/files/ftp_upload/1115/Figure%203%20FINAL.png" alt="" /> 图3，在受精后24小时，uninjected的胚胎有一个长期的，直身无坏死或中线（A）和（二）之间的神经管缺陷的两个明确的眼睛。 HEG的mRNA注入一些胚胎，相反，表现出缩短身体的纵轴，坏死，misshaped体节（三），和独眼畸形（四）。 （A）和（C）是4X DIC的图像;（B）和（d）为20倍DIC的图像。

<ol>
	<li>Mably, J. D., Mohideen, M. P. K., Burns, C. G., Chen, J., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heart+of+glass+regulates+the+concentric+growth+of+the+heart+in+zebrafish.">heart of glass regulates the concentric growth of the heart in zebrafish.</a> Curr. Biol. 13, 2138-2147 (2003).</li></ol>

斑马鱼模型系统的优势之一是与特定的基因产物可以添加或从胚胎显微注射所淘汰的缓解。无所不在的过度表达一种特定的蛋白质，mRNA编码是注入蛋黄1细胞阶段。在胚胎的后续发展，RNA是分布在整个生物体和翻译。相反，要消除一种特定的蛋白质，morpholinos使用。 Morpholinos是与特定的RNA的反义互补性，设计合成寡核苷酸。一样的mRNA，morpholinos注入蛋黄1细胞阶段。在胚胎里，他们结合自己的靶RNA，防止翻译。 主要的修改，需要为每个啉或mRNA注射材料的浓度。过高的浓度啉或表达可能导致非特异性的毒性，而每个啉必须的实证检验，以确定其最佳浓度，通常啉浓度200微米，500微米之间的基因的活动有效地击倒，而不会造成非特异性缺陷。针开放精确的尺寸并不重要。针头大小的范围内，注射压力和注射时间内可以进行调整，以产生正确的卷丸。 Morpholinos有许多应用，包括域范围内的一种蛋白质的功能夹层。设计针对特定的外显子 - 内含子路口时，morpholinos防止发生有拼接。我们研究了两个morpholinos的影响，结合外显子-内含子在mRNA前体的 HEG的交界处。啉heg_e3i3_egfr1结合的第3外显子和内含子3之间的交界处，防止纳入成熟笔录第3外显子剪接机器。吗啉heg_e4i4_egfr2同样删除第4外显子。外显子3和4都含有EGF样重复编码域。有些胚胎注入heg_e4i4_egfr2中等型模拟突变，将需要进一步的实验，以了解HEG第4外显子在心脏发育中的作用。令人惊讶的是，与heg_e3i3_egfr1注射的胚胎脑水肿，HEG突变体的功能。这可能是由于morpholinos能力结合产妇的成绩单将在合子突变体的影响。

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<td>Sutter Instruments</td>
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<td>World Precision Instruments</td>
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microinjection of zebrafish embryos to analyze gene function

研究斑马鱼的优点之一是操纵在胚胎中的蛋白质水平的易用性和速度。 Morpholinos，这是与靶RNA的合成寡核苷酸反义互补，可以添加到胚胎，以减少一个特定的基因产物的表达。相反，处理的mRNA可以添加到胚胎，增加一个基因产物的水平。车辆无论是基因或吗啉胚胎显微注射。显微注射是高效，快速，让数百个胚胎每小时注入。该视频显示在显微注射的所有步骤。简单地说，鸡蛋收集后立即被解雇，一字排开反对在培养皿中的显微镜幻灯片。下一步，注射材料装入了一种细尖的针是连接到一个微量空气源，并微量控制的调整，以产生一个理想的注射量。最后，针陷入胚胎的卵黄和吗啉或mRNA被驱逐。

Watch this Scientific Journal Video about Microinjection of Zebrafish Embryos to Analyze Gene Function at JoVE.com

Microinjection of Zebrafish Embryos to Analyze Gene Function

该视频演示如何啉或mRNA可以注射到斑马鱼的胚胎，在单细胞阶段减少或增加特定的基因产物在随后的发展水平。

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 ...

microinjection-of-zebrafish-embryos-to-analyze-gene-function

Research

JoVE Journal

Biology

77.2K 次观看.  Harvard Medical School. 该视频演示如何啉或mRNA可以注射到斑马鱼的胚胎，在单细胞阶段减少或增加特定的基因产物在随后的发展水平。

视频: Microinjection of Zebrafish Embryos to Analyze Gene Function

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 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 ...

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 ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

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 ...

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 ...

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

基因捕获斑马鱼中使用的Gal4

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...