这项工作是由美国国家科学基金会的IOS 0844948和1024811的IOS支持

保留所有海水或人工海水（SW），成年动物，并且培养在15℃之多是实用的。确保鸡蛋和受精卵被浸在西南。 商业准备海盐再造用蒸馏水或反渗透水供应以及西南的来源。检查盐度用比重计，调整盐或水，以达到最佳的水平。保持1.020之间比重水平1.025。保留所有玻璃器皿和其他塑料制品实验室器皿分开，以避免化学物质的污染。清洁胚胎级实验室器具通过使用去离子水或稀次氯酸钠随后在水漂洗几次偶尔浸泡漂洗。 从Patiria君子兰成人1获取和成熟配子<ol><li>选择一种动物来切除性腺。通过寻找卵巢或睾丸的存在决定切除术后性生活，因为体育君子兰是不是本身xually二形。 </li><li>切一个小口，不超过1厘米时，沿海星手臂一边用手术刀片（ 图1A）。使用小钝端钳子拉回切口的边缘以访问性腺组织和拉性腺组织通过所述切口。 注意:避免拉出内脏组织，这是一个松散的灰色或褐色组织（ 图1B）。卵巢通常是橙色，黄色或浅棕色的（ 图1C），同时睾丸是白色或米色和当中断将导致海水成为在外观上（ 图1D）乳状或浑浊。 </li><li>回到女性的水族馆。隔离男性的一两个小时在西南的容器，因为从处理压力可能诱发产卵。动物愈合沿切口部位，并继续提供配子几个月。 </li><li>收集的睾丸到1.5ml的Eppendorf管中以尽可能少的SW尽可能并在冰上储存直至使用。存储任何睾丸不是我们版上收集的当天，在4℃下长达一个星期。 </li><li>收集在一个小的玻璃培养皿卵巢在西南的几毫升。从卵巢组织释放卵母细胞，不是分裂组织有两对钳，直到没有大块依然存在。 注:最佳地，大部分的卵母细胞被充分开发。这样的卵母细胞是大的，圆的，并且澄清的黄色或浅棕色，在卵母细胞（ 图2A），一个独特的胚泡，相比于未开发的卵母细胞的体积更小，褐色，呈粒状，且不透明的（ 图2B）的子集。如果高于25％左右的卵母细胞是未开发的品种，选择不同的女性获得的卵母细胞。 </li><li>倒入通过网状过滤杯的卵母细胞和组织剩余使用200微米的孔径，收集流过。这将包括卵母细胞的所有品种，但去除卵巢组织。从捕获过滤器通过喷射瓦特组织打跑剩下的卵母细胞从喷瓶第i西南。滤杯使用的是50ml锥形管（ 图3A-A"）进行。 </li><li>通过倒入步骤1.6流通过100微米筛网过滤杯。舍弃小的卵母细胞在流过。大充分发展的卵母细胞是准备成熟会留在过滤器上。冲洗的卵母细胞到一个干净的200毫升烧杯中。 </li><li>添加95毫升SW向卵母细胞中的200毫升玻璃烧杯中。加入5 ml的200μM1 - 甲基腺嘌呤为10μM的终浓度。 </li><li>转移的卵母细胞中，以大量浅培养皿中并置于在15℃。高的表面积与体积培养皿允许氧气交换。卵母细胞并不成熟，也可以不发展好，如果他们过于拥挤在这个成熟的阶段。分成西南几盘加10 M 1-methlyadenine，直到文化是小于200的卵母细胞/毫升。 </li><li>允许的卵母细胞成熟为45-90分钟，但不再因为这将使得无法成为卵母细胞˚Fertilized。要确定是否成熟是完整的，看的卵母细胞在解剖范围。对胚泡移动和熔断器与细胞膜成熟过程。然后将其分解，因此不再在成熟的卵母细胞（ 图2C）中可见。 </li></ol> 2，施肥成熟的卵母细胞<ol><li>在一个小的玻璃培养皿约1毫升的SW选择了一小簇睾丸组织中，大约豌豆大小，与剁碎的。 </li><li>用巴斯德吸管2-3滴生成的乳白色海水转移到大约1​​00毫升的成熟卵母细胞和晃动培养皿混匀。避免传输睾丸组织碎片。避免增加更大量的精子，因为这可能会导致多精受精和随后的扶贫开发。 </li><li>几分钟后观察下解剖范围的卵母细胞。受精卵，或合子，有施肥的信封，这是一个明显的泡沫升起关闭CEL升膜（ 图2D）。 注意:不要进行培养，如果低于90％左右的卵子受精，因为可怜的受精率是一种不健康的文化，可能还没有发展很好的指示。 </li><li>受精完成后，通过100μm的筛网过滤器浇注和漂洗入培养皿以新鲜SW改变对受精卵西南。它除去过量的精子，以防止氧饥饿是很重要的。将培养皿在15℃下15-30分钟。 </li></ol> 3，德jellying和划船受精卵<ol><li>通过从60×15毫米聚苯乙烯培养皿取盖准备注入的菜肴。 <ol><li>画上的菜的后面，周围的显微注射显微镜相匹配的视野面积永久性记号笔线。这确保了所有棱胚胎视显微镜视野内。 </li><li>使用玻璃巴斯德吸管穿过圆P的得分线referably关闭的圆（ 图3B）的一侧上。使用此线后，打破注射针头（步骤4.5）。 注:试剂通常用于粘附海胆胚胎， 如硫酸鱼精蛋白或聚-L赖氨酸，是不必要的。德胶状体育君子兰受精卵坚持得非常好无涂层塑料。需要注意的是，他们不坚持好玻璃餐具。 </li></ol></li><li>添加周围10毫升SW每个培养皿，使得表面被覆盖，但水不会过于飞溅的菜，因为这可能会扰乱棱胚胎。 </li><li>除去体育君子兰受精卵果冻大衣搭配酸申银万国之前，赛艇。这果冻大衣比观察模型海胆种类更加广泛。 <ol><li>制备酸SW在pH值4.0-4.2的范围内。 pH值是重要的，以允许有效地除去透明带的同时允许正常发育。 </li><li>在西南添加单滴1 N（1米），盐酸申银万国的1升的股票。让每一滴完全混合和加入更多的HCl之前监测pH值。 注意:一至几毫升的盐酸结果在适当的pH值，但不一次性添加的整个体积。 注:准备1N的HCl的通风柜与SW和12 N（12米）的HCl。 </li><li>通过浇注他们到100微米的网眼过滤器上的受精卵中取出西南。冲洗至200ml烧杯中，在西南可能的（约10毫升）的最小量。填充该烧杯高达150毫升酸SW（pH为4.0）和允许的受精卵坐在酸SW 3分钟。 </li></ol></li><li>也就是把它们来回两个200毫升的烧杯中的果冻剥去外衣从受精卵，经过200微米的筛网过滤过的受精卵各一次。通过5到10倍左右的过滤器在大约2分钟的时间窗口倒受精卵。 </li><li>浇受精卵到100微米的网眼过滤器除去酸西南。冲洗受精卵到一个小的玻璃培养皿西南。德果冻受精卵可能会粘到塑料餐具。立即排胚胎;他们开始产生更多Ĵ伊丽大衣从而导致粘合性差的塑料餐具随着时间的推移。 </li><li>用移液管嘴（ 图3C-C&#39;）接受精卵从玻璃培养皿中，并将它们插入注射菜肴直线。熔融巴斯德吸管的薄侧的中心在火焰直到玻璃是软的。快速拉玻璃板的端部开以产生非常薄的拉伸玻璃。一旦玻璃很酷，撅着巴斯德吸管的小端（如需有关准备口吸管看到Wessel 等人 ，2004年17或甜等人 ，2004年18）。 <ol><li>做一个划船吸管使得直径只比胚胎的直径大，这样只有一个受精卵会进入和退出吸管的时间。这将允许单排形成。较小直径的吸管可以剥离包膜肥料，破坏受精卵为P.君子兰受精卵没有一个透明膜上第二是比较软。 </li><li>如果受精卵没有坚持到塑料盘，让他们有更多的时间在酸性申银万国（可达数多钟）。另外一个更小直径的吸管可以帮助去除透明带，使受精卵能够更有效地坚持下去。 </li><li>受精卵略有正浮力，而且往往浮动或只是悬停在培养皿底部上方。在这种情况下，使受精卵被轻轻地压在它们离开吸管（ 图3D）的培养皿的表面定位划船吸管。 注:这是允许的，以适应这些菜多P.行君子兰胚胎膜不会变得更难以穿透时间的推移，因此可以注入数百受精卵在单个盘。 </li></ol></li></ol> 4，注射受精卵<ol><li>在终浓度准备注射液适当浓度的吗啉反义寡核苷酸（MASO），基因或DNA的entration 200毫米氯化钾。 400-800微米MASO和DNA的2.5纳克/微升往往会产生morphant表型，同时最大限度地减少毒性。在蛋中的最终浓度将是125,1 个起始浓度14。对于含有MASOs，热至65℃5分钟，注射溶液来使用，然后储存在​​室温下立即之前。 <ol><li>为了便于排序注射的胚胎中，添加罗丹明葡聚糖示踪剂的注射溶液，以获得0.1％的浓度。 </li></ol></li><li>通过在针拉仪器依照制造商的指示拉玻璃毛细管（1.0 mm外径×0.75毫米内径，100mm长）制备注射针头。适用于注射针头的海胆同样适用于海星，所以用类似的参数19。 </li><li>装入约2微升注射液为使用microloader枪头注射针。 </li><li>插入针的钝端插入米anipulator。设置picospritzer到100毫秒的脉冲，并在40 PSI的空气压力。 </li><li>将划胚胎的菜到显微镜载物台，并拥有所有胚胎的视野。定向的盘，使得刻痕是在最靠近针头的侧面。定位针，在一个大约60°角，以便尖端附近的线的中间，并且只在水表面上方。 </li><li>着眼于刻划线和降低针，直到针尖进入焦点。降低尖一点，打破它轻轻跑入得分线的脊开放。提高尖断菜的表面，并在受精卵的第一行的顶部位置，并专注于受精卵。 </li><li>降低尖，这样它会刺穿合子的中心周围60度角进入。针很容易穿透体育君子兰受精卵。踩脚踏板（或者迫使材料从注射针的其它装置）上引入仁济的大丸剂ction溶液进胚胎。 </li><li>瞄准丸剂是20-30％的胚胎的直径，或80之间25和PL。如果推注比这个更大或更小，调整注射的picospritzer的持续时间和注入下一个胚胎。继续调整的持续时间，以获得所需丸剂的大小。开始在100毫秒时间。如果大于大约200毫秒，需要引入相应的弹丸重断针。丢弃的针，如果小于15-20毫秒，才能获得这个弹丸大小的针头大小过大，可能打乱正常的发展作好准备一个新的。 </li><li>继续注入剩余的受精卵在注射菜。胚胎可以容易地注入到切割开始和到第一切割后单卵裂球。定期提起水的表面上方的针以除去粘附在其针合子。如果有必要重新打破针的前端，或如果发生堵塞时加载新的针。 </li></ol><ol><li>让胚胎在15℃培养在西南。保持高的表面积与体积的培养物，以允许氧气交换。确保胚胎不拥挤;保持培养物在等于或低于200个胚/毫升。根据胚胎的期望阶段，计划收集注射的胚胎在约20-24小时后受精。 注:这通常是一个理想的时间整理和收集，因为发育正常的胚胎很容易区分形态，但尚未游泳。 </li><li>如果胚胎孵化，轻轻盐冲击，防止游泳。加入200μl5 M氯化钠10毫升的SW在注射盘中并轻轻摇动使西南。几分钟后，将胚胎将下降到培养皿的底部。收集这些，并将其移动到正常盐度西南。 </li><li>收集在一个合适的荧光光源在injecti使用的示踪剂兼容注射的胚胎在解决方案。如果未使用的示踪剂，排序的胚胎以选择正常的形态。 </li><li>用口吸管，吸取荧光的胚胎与西南的少量吹出来到一个小培养皿中充满了西南。理想口移液管具有足够大的开口，用于胚胎进出被拉而不损坏它们，但没有大到不希望的胚和外来SW也拉升。 </li><li>一旦所有的所需胚胎已收集到新的培养皿，观察在荧光下的胚胎和除去任何未注射的胚胎或发育不良的胚胎。 </li><li>文化分拣胚胎所需的阶段，孵化器，并进行影像或其他所需的协议。 </li></ol>

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<li>Hinman, V. F., Nguyen, A., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caught+in+the+evolutionary+act%3A+precise+cis-regulatory+basis+of+difference+in+the+organization+of+gene+networks+of+sea+stars+and+sea+urchins%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Caught in the evolutionary act: precise cis-regulatory basis of difference in the organization of gene networks of sea stars and sea urchins.</a> Dev. Biol. 312, 584-595 (2007).</li>
<li>McCauley, B. S., Weideman, E. P., Hinman, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+conserved+gene+regulatory+network+subcircuit+drives+different+developmental+fates+in+the+vegetal+pole+of+highly+divergent+echinoderm+embryos%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">A conserved gene regulatory network subcircuit drives different developmental fates in the vegetal pole of highly divergent echinoderm embryos.</a> Dev. Biol. 340, 200-208 (2010).</li>
<li>Wessel, G. M., Voronina, E., Brooks, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obtaining+and+handling+echinoderm+oocytes%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Obtaining and handling echinoderm oocytes.</a> Methods Cell Biol. 74, 87-114 (2004).</li>
<li>Sweet, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blastomere+isolation+and+transplantation%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Blastomere isolation and transplantation.</a> Methods Cell Biol. 74, 243-271 (2004).</li>
<li>Cheers, M. S., Ettensohn, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+microinjection+of+fertilized+eggs%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Rapid microinjection of fertilized eggs.</a> Methods Cell Biol. 74, 287-310 (2004).</li>
<li>Core, A. B., Reyna, A. E., Conaway, E. A., Bradham, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pantropic+retroviruses+as+a+transduction+tool+for+sea+urchin+embryos%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Pantropic retroviruses as a transduction tool for sea urchin embryos.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 5334-5339 (2012).</li>
</ol>

没有利益冲突的作者声明。

但是也有一些困难，这种技术的初学者，但对于成功创建morphant胚胎必不可少的两个关键步骤。首先是选择健康的卵母细胞，将成熟和受精正常。正常发展的一种文化的百分比取决于季节，动物的健康，以及卵母细胞已收获从单一个体的次数。卵母细胞往往是从四月到十月质量更好。仔细看看卵母细胞着手确保广大看起来像图2A，而不是图2B以前是很重要的。但也允许有少...

海星，Patiria君子兰 ，（俗称蝙蝠星）正在成为一个有趣的和通用的模型系统，适用于各种细胞1-3，4,5发育，进化6-8和生态研究9-11。成人体育君子兰分布以及从锡特卡，阿拉斯加到巴哈加利福尼亚州12太平洋沿岸和海洋水族馆很容易维护。卵母细胞获得全年每一位女性能够摆脱鸡蛋数以万计。卵母细胞容易成熟，受精外部13。由此产生的胚胎是透明的，允许，便于观察;他们同步发展，并且只需要海水的发展。全基因组组装和多个转录，也可用于P。君子兰 （ <a href="http://www.echinobase.org/Echinobase/">Echinobase.org</a> ）。这些优点使其非常适合各种研究与教学之用。 近年来，P.君子兰已成为发育的基因调控网络模型系统分析14-16。这些研究的目的是确定调节基因的整个称赞，并确定它们之间的相互作用的网络。这项工作的，需要通过引入反义寡核苷酸或体外合成的mRNA扰动的基因表达。此外， 顺式调控分析用于表征调节DNA 15的功能。这些分析需要引入扰动试剂和/或DNA报道构建成胚胎。此外，以表征这些扰动的下游作用，必须测定许多胚胎的变化的潜在靶基因的表达。对于数百受精卵的显微注射技术是核心对这项工作。 棘皮类动物，其中包括体育君子兰 </em&gt;的，需要很多个月才能达到性成熟。因为这一点，所以一般不实用，开发和维护用于实验的转基因株系这些动物。因此，转基因的成年人育种不能有效地创建改性胚胎。相反，扰动必须发生从头通过显微注射。注射提供了一个机会来修改胚胎试剂不属于细胞渗透性。以下协议描述的方法引入DNA，基因，细胞示踪和吗啉反义寡核苷酸为数百个受精卵在一个2-3小时坐通过显微注射。这将产生足够的材料，适用于各种下游实验包括，但不限于，定量PCR， 原位杂交，RNA测序和Western印迹。

<table><tbody><tr><td>1-Methyladenine</td><td>Acros Organics (Fisher Scientific)</td><td>AC20131-1000</td><td></td></tr><tr><td>190 micron nitex nylon filter</td><td>Small Parts (originally Sefar)</td><td>CMN-0185-C/5PK-05</td><td></td></tr><tr><td>100 micron nitex nylon filter</td><td>Small Parts (originally Sefar)</td><td>CMN-0105-C/5PK-05</td><td></td></tr><tr><td>Polystyrene Petri dishes, 60 mm x 15 mm</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Capillary tubing</td><td>FHC, Inc</td><td>30-30-0</td><td>For pulling microinjection needles</td></tr><tr><td>Model P-97 Needle Puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Dextran, Rhodamine Green</td><td>Life Technologies</td><td>D7163</td><td>If injecting a GFP expression reporter, it is helpful to substitute Texas Red dextran as an injection tracer</td></tr><tr><td>Instant Ocean Sea Salt</td><td>Doctors Foster and Smith</td><td>CD-116528</td><td>Also available in many pet stores</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242 956.003</td><td></td></tr></tbody></table>

a method for microinjection of patiria minata zygotes

棘皮动物，长期以来就是繁殖和发展研究的最喜爱的模型系统，以及最近的基因调控和发育过程的演化的研究。海星，Patiria君子兰 ，是获得流行为这些类型的研究而以前进行几乎完全的海胆， 海胆紫石和Lytechinus斑的模型系统。这些模型系统的优点是易于制备改性胚胎中的特定基因或下调，标记细胞的一个群体，或引入报告基因。单一显微注射方法能够创建各种这样的修改胚胎的。在这里，我们提出了一个方法，从体育取得配子君子兰 ，产生受精卵，并经由显微注射引入扰动的试剂。健康morphant胚随后隔离GE的定量和定性研究NE功能。基因组和转录组数据，这个​​有机体的可用性增加了执行和便于执行​​它们的研究类型。

该协议的目标是引进试剂成胚胎。我们证明了通过注射的DNA报告构建，推动绿色荧光蛋白（GFP）表达的协议的有效性。注射的胚胎表达GFP的克隆斑块（ 图4A-B），是将DNA中的早期卵裂结合。许多试剂是希望引进胚胎在高数量和胚胎的次优批次有毒。毒性舱单，延迟发展，阻止发展中的早期卵裂或在受精卵阶段，或通过施加异常发展（ 图5A-C）。 <p class="jove_content" fo:keep-t...

Watch this Scientific Journal Video about A Method for Microinjection of Patiria minata Zygotes at JoVE.com

A Method for Microinjection of Patiria minata Zygotes

产生morphant胚胎的方法是必不可少的学习发展机制和基因调控网络。海星Patiria君子兰是这些研究的一个新兴的模型系统。在这里，我们提出了一个协议获得配子，胚胎生产的文化，并从该植物受精卵显微注射迅速。

用于显微注射的方法<em&gt; Patiria minata</em&gt;合子

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

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Research

JoVE Journal

Biology

A Method for Microinjection of Patiria minata Zygotes

11.0K 次观看.  Carnegie Mellon University. 产生morphant胚胎的方法是必不可少的学习发展机制和基因调控网络。海星Patiria君子兰是这些研究的一个新兴的模型系统。在这里，我们提出了一个协议获得配子，胚胎生产的文化，并从该植物受精卵显微注射迅速。 

视频: A Method for Microinjection of Patiria minata Zygotes

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

科研

遗传学

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 ...

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 ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

发育生物学

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

冈 比亚按蚊 胚胎的显微注射方法

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

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 ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Zebrafish Microinjection Techniques

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development.
This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA injection, gene silencing by delivery of antisense morpholino oligonucleotides, and the generation of transgenic zebrafish using specially engineered plasmid DNA.

Introduction to Microinjection of Early Zebrafish Embryos

斑马鱼显微注射技术

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage ...

用于显微注射的方法

摘要

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激光辅助畜禽受精卵细胞质显微注射

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

斑马鱼的胚胎显微注射来分析基因的功能

斑马鱼幼虫显微注射和成像的微装置

冈比亚按蚊胚胎的显微注射方法

作为一个型号的遗传生物鳉胚胎显微注射

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus

海胆受精卵的高通量显微注射

斑马鱼显微注射技术

用于显微注射的方法

摘要

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激光辅助畜禽受精卵细胞质显微注射

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斑马鱼的胚胎显微注射来分析基因的功能

斑马鱼幼虫显微注射和成像的微装置

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作为一个型号的遗传生物鳉胚胎显微注射

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus

海胆受精卵的高通量显微注射

斑马鱼显微注射技术

冈比亚按蚊胚胎的显微注射方法