这项工作是从MRC的赠款支持。

1。交配和收集鸡蛋<ol><li>女性和男性鳉可以很容易地分辨臀鳍和背鳍的大小和形状。的女性相比，男性的臀鳍较大，平行四边形形。女性臀鳍较小，三角型。男性的背鳍之间的最后两条射线清晰可见深的缺口。 </li><li>鳉奠定了每天40鸡蛋和青鳉女性进行聚集在他们肚子里几个小时的附件细丝的鸡蛋才剥离在水箱中的植物。 </li><li>女性可以捕净和举行内部两侧轻轻一方面。鸡蛋然后可以从另一方面食指腹轻轻地戏弄。女性可以再返回油箱。鸡蛋应转移到与胚胎媒体充满钝钳或手指一个6厘米的培养皿。 </li><li>鳉对很少打时，他们都保持在一个小交配框。因此，他们可以在一个小坦克与水流，使他们可以喂养和鸡蛋可以收集几个月每天从同一对。 </li><li>重要的是母鱼不应该保持无男性，因为排卵发生在鳉女性的日常。否则，女性不能提供鸡蛋，成为不适。 </li></ol> 2。清洁胚胎<ol><li>转让非集群鸡蛋一个玻璃吸管放置在一个9厘米培养皿的盖子，以砂纸（P2000粒度，防水）。删除多余的介质，但保证足够的量仍然以防止干燥胚胎。 </li><li>砂纸使用的食指轻轻地滚上的胚胎，用最小的压力，并保持了大约45-60秒手指砂纸表面平行，以消除一些绒毛膜外表面毛。滚不要超过一次5-7胚胎此预防措施，将最大限度地减少风险破碎下对方的胚胎。 </li><li>清洗的胚胎转移到胚胎培养基的新鲜菜。 </li></ol> 3。期间举行显微注射​​胚胎琼脂糖板在与模具（图1）在1.5％琼脂糖有机玻璃制成的注射，胚胎被关押在低谷。琼脂糖的浓度是很重要的妥善举行胚胎。 <ol><li>首先，琼脂糖帧举行的有机玻璃模具。 1.5％琼脂糖倒入水在一个9厘米培养皿0.5厘米深度和等待，直到它完全凝固。切出模具的大小仅比大面积琼脂糖。 </li><li>倒入1.5％的琼脂糖填补截面积和地点，无气泡，使被困模具，等到琼脂糖凝固。这琼脂糖板可存放在冰箱加快solidifaction。 </li><li>取出模具创建槽举行的鸡蛋。补充足够的胚胎中期沉浸在4℃直至所需的模具，封面和存储。 <img src="/files/ftp_upload/1937/1937fig1.jpg" alt="图1" /> 图1。定做模具用来准备在显微注射琼脂糖板槽的示意图。 </li></ol> 4。青鳉胚胎微量注射在青鳉DNA，RNA，吗啉代寡核苷酸和示踪染料是显微注射绒毛膜进入细胞质中的1到64细胞阶段的胚胎，而不是在斑马鱼的卵黄。由于细胞质在青鳉更小，更可见，注射染料如罗丹明-葡聚糖是建议制定这个技能的做法。酚红作为示踪此外还鼓励，允许注射的确认。 由于周围的青鳉胚胎绒毛膜是艰难的，一个短，结实，宽的注射针（图2）。准备从灯丝含玻璃毛细管这种强烈的广泛的注射针头。 <ol><li>准备注射液与酚红作为示踪剂，并与Eppendorf公司microloader背面装载进针。空气泵喷油器的针连接。 </li><li>鸡蛋必须尽快收集和非集群受精后注入单细胞阶段。可以注入更多的胚胎，它们储存在冰（不超过30分钟），阻止细胞分裂。 <img src="/files/ftp_upload/1937/1937fig2.jpg" alt="图2" /> 图2。鳉和斑马鱼针的比较。注意的MF针尖（顶）更短和更广泛的，因此鳉胚胎周围的强硬绒毛膜穿刺。 </li><li>琼脂糖板转移到鸡蛋和调整钳波谷。注射前，5个鸡蛋，应该是这样的一个细胞的细胞质的细胞膜可以打日的旋转Ë针直刺。一个细胞的细胞质中可以发现，寻找一个没有面积小的油滴，这是对面一侧的鸡蛋最密集的油滴。从侧面观看，所确定的区域可以被证实为细胞质。 </li><li>将针头靠近鸡蛋。打开针尖轻轻触摸针尖绒毛膜或使用microforceps（图3）。应该看到少量的注射物质流出来了。 </li><li>通过绒毛膜穿刺放置绒毛膜针内的一角。调整喷油器的控股是比较高的，以确保没有回流的发生是由于穿刺后高的压力，通过艰难的绒毛膜（80 100hPA设置保压压力和注射压力500 - 700hPA）的压力。 </li><li>通过大幅戳其膜，进入细胞质中插入的针尖。 避免针尖插入蛋黄。不同的斑马鱼，注入蛋黄的材料将不被转移到细胞质中。注入的液体有明显的边界时注入蛋黄，而边界是模糊的，当在细胞质中（图4）。 偶尔针可能成为阻止琼脂糖/碎片。镊子可用于触摸/ rebreak针年底，清除堵塞。如果针在注射过程中的任何一点断裂，鼓泡将被视为胚胎培养基中，你是注射的物质都将丢失。注射剂应进行系统从底部沿琼脂糖渠道，由左向右横跨模具。 </li><li>注射的胚胎被转移到一个干净的菜，含有胚胎培养基，并在适当的温度开发。 <img src="/files/ftp_upload/1937/1937fig3.jpg" alt="图3" /> 图3。断裂的针头注射前。 1：移动提示针接近胚胎绒毛膜; 2：触摸绒毛膜和向前和向后移动针轻轻打破头; 3：当少量的物质注射针年底流动确认破损小费。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/1937/1937fig4.jpg" alt="图4" /> 图4。注入青鳉胚胎的代表图像。小组A显示了一个正确罗丹明-葡聚糖注射到一个细胞阶段鳉胚胎的细胞质的形象代表。小组B显示了一个不正确的注入一细胞阶段的青鳉胚胎的卵黄囊罗丹明-葡聚糖的形象代表。面板C和D显示面板的胚胎，A和B，分别在注射后约30小时。 EM：胚胎，前面板中向上的C和D，并查看背。请注意面板ð胚胎身正，不红染料被错误地注入到卵黄囊。在B和D的箭头指示罗丹明-葡聚糖注入卵黄囊不正确，请注意不同的圆形边界。点分线A和B表明，单细胞的细胞质的轮廓，视图是横向的。

<ol>
	<li>Furutani-Seiki, M., Sasado, T., Morinaga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genome-wide+screen+for+mutations+affecting+organogenesis+in+Medaka,+Oryzias+latipes.">A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.</a> Mech Dev. 121, 647-658 (2004).</li><li>Furutani-Seiki, M., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka+and+zebrafish,+an+evolutionary+twin+study.">Medaka and zebrafish, an evolutionary twin study.</a> Mech. Dev. 121, 629-6237 (2004).</li></ol>

在这段视频中，我们展示了一细胞阶段鳉胚胎细胞示踪，基因，DNA或反义寡核苷酸注射的方法。这项技术已经允许我们研究各种发育过程实时在体内 。这个过程和随后的详细分析，它可以提供有可能显着提高我们的脊椎动物器官和细胞生物学的基本认识。这种知​​识可能是重要的，产量在再生医学治疗应用。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>4" fine net</td>
<td>Tools</td>
<td>J &amp; K Aquatics Ltd.</td>
<td>MD001</td>
<td>Used to capture fish during egg harvesting.</td>
</tr>
<tr>
<td>Embryo medium</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>&nbsp;</td>
<td>200ml 50X stock solution, 1ml 1%methyleneblue in H2O/10 liter RO water (50X stock solution: NaCl 14.7g, KCl 0.6g, CaCl2 2H2O 2.4g, MgSO4 7H2O 4.0g/1 litre RO water.</td>
</tr>
<tr>
<td>Fine waterproof sandpaper - p2000 grit size </td>
<td>Tools</td>
<td>Hermes Abrasives Ltd.</td>
<td>&nbsp;</td>
<td>Very fine grit size for rolling/cleaning embryos with chorions.</td>
</tr>
<tr>
<td>Borosilicate glass capillaries</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>GC100-10F</td>
<td>For making microinjection needles.</td>
</tr>
<tr>
<td>Micropipette puller</td>
<td>Equipment</td>
<td>Narishige scientific instrument lab</td>
<td>Model PP-830</td>
<td>For making microinjection needles.</td>
</tr>
<tr>
<td>Eppendorf microloader</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>5242956.003</td>
<td>For loading microinjection needles.</td>
</tr>
<tr>
<td>Microinjector apparatus </td>
<td>Equipment</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>Model 5242.</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Equipment</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>Model MN151.</td>
</tr>
<tr>
<td>Agarose injection plate</td>
<td>Tools</td>
<td>Made in-house</td>
<td>&nbsp;</td>
<td>For holding embryos during injection.</td>
</tr>		</tbody>
		</table>
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microinjection of medaka embryos for use as a model genetic organism

在这段视频中，我们展示了一细胞阶段鳉胚胎显微注射技术。青鳉是一个小产蛋的淡水鱼类，允许遗传和胚胎学分析，是脊椎动物的模式生物体的表型驱动的全基因组突变的屏幕进行了1在斑马鱼和小鼠，之一。鳉和斑马鱼之间的相关基因的功能重叠的分歧使得新颖，品种单一，从而鳉和斑马鱼是脊椎动物的基因组功能基因清扫互补难以辨认的表型鉴定。 为了充分利用鳉鱼的胚胎是透明的，发展的外部，显微注射法是一个重要的技术注入基因过度表达，敲下来的利益和国家主管部门作出标签的细胞，mRNA的反义寡核苷酸细胞示踪转基因株系。

Watch this Scientific Journal Video about Microinjection of Medaka Embryos for use as a Model Genetic Organism at JoVE.com

Microinjection of Medaka Embryos for use as a Model Genetic Organism

鳉和斑马鱼是脊椎动物的基因组功能基因夹层互补。该协议凸显成功鳉胚胎微量注射，在实验室中使用的青鳉和斑马鱼的胚胎和遗传分析的重要技术关键点。

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 ...

microinjection-of-medaka-embryos-for-use-as-a-model-genetic-organism

Research

JoVE Journal

Biology

25.5K 次观看.  University of Bath. 鳉和斑马鱼是脊椎动物的基因组功能基因夹层互补。该协议凸显成功鳉胚胎微量注射，在实验室中使用的青鳉和斑马鱼的胚胎和遗传分析的重要技术关键点。

视频: Microinjection of Medaka Embryos for use as a Model Genetic Organism

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

霍乱弧菌：模式生物研究细菌的发病机制 - 访

科研

生物学

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 ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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 ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. 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 the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...