Name: sand fly (phlebotomus papatasi) embryo microinjection for crispr/cas9 mutagenesis
Uploaded: 2020-11-17T15:00:00.000+00:00
Duration: 5 min 44 s
Description: Watch this Scientific Journal Video about Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis at JoVE.com

作者感谢瓦妮莎·梅尔德纳-哈雷尔对手稿的批判性解读。

严格按照《国家卫生研究院实验室动物护理和使用指南》的建议，使用小鼠作为沙蝇喂养的血液来源。该议定书得到了国家艾滋病规划署动物护理和使用委员会（协议编号LPD 68E）的批准。无脊椎动物不在国家卫生研究院指南的涵盖范围之内。
1. 针头制备 （图1）
<ol><li>拉针和斜面，如在梅蒂和哈雷尔19描述。简言之，使用薄氧酸玻璃毛细管在双级玻璃微皮拉器上拉针。</li>
	<li>用一块额外的细石，在微尖斜面上用湿斜面切针。</li>
</ol>2. 胚胎收集和微操纵 （图2）
<ol><li>注射前五天，血液喂养沙蝇雌性，定期进行菌落维护。有关沙蝇群落饲养程序和所需材料的详细信息，请参阅律师等人1 在整个开发过程中，在 70% 湿度和 26 °C 的孵化器中保持昆虫。<ol>
<li>有一天，在喂血后，用石膏锅和侧端口的100-150组女性捕捉喂血的女性。通过用糖溶液（30%蔗糖-足够的体积浸泡一个小棉球）喂养雌性，这是通常的殖民地维护。</li>
	</ol>
</li>
	<li>在第2-3天献血后，不要滋润石膏锅。将雌性放在干燥的基材上可以防止过早产卵，因为雌性更喜欢在潮湿的环境中产卵。</li>
	<li>第5天进行血液喂养后，进行胚胎采集、微操纵和微注射。将湿润滤纸引入产卵室。新鲜铺设的胚胎没有完全发育的胆汁，呈白色。在手术过程中使用黑色滤纸，以促进白色胚胎的可视化。</li>
	<li>使用口腔吸气器将一小群女性从石膏锅转移到产卵室，通过侧端口。湿润基板（滤纸）的存在诱使女性渗出。</li>
	<li>30-60分钟后，小心地从腔室中取出新铺设的胚胎的滤纸。将滤纸和胚胎放在培养皿中长达3小时。在此期间，定期评估滤纸的水分水平，以确保其保持湿润。在此期间，重复步骤2.4至2.6与新的女性群体，并收集尽可能多的胚胎。</li>
	<li>准备显微镜幻灯片进行注射:用非常精细的画笔手动收集鸡蛋，并小心地将它们转移到另一张湿润的黑色滤纸上，放在显微镜滑梯上，上面有玻璃盖片。<ol>
<li>将水加入滤纸中，足以保持胚胎湿润，但不会使胚胎从盖片上浮出或被吸到盖片下。将胚胎与盖片对接。盖片起到后挡作用，防止卵子在注射过程中滚走。</li>
	</ol>
</li>
</ol>3. 胚胎注射 （图3）
<ol><li>鸡蛋收集开始后 2.5 至 3 小时开始注射。在室温和环境实验室湿度下进行注射。</li>
	<li>确保对齐的胚胎有足够的水，使其保持湿润。 注:在注射过程中，胚胎必须保持湿润。如果注射过程中没有湿润，注射变得困难，因为针头难以刺穿胚胎。正确的水量是胚胎周围有半月板水的点，但盖片不会漂浮在水面上，导致胚胎在盖片下推。在注射过程中监测胚胎周围的水量至关重要。根据需要加水以保持胚胎湿润。</li>
	<li>用水润湿刷子并将水转移到滤纸的后端，小心地加水。以这种方式添加水可以以缓慢和可控的方式添加水，以便添加足够的水来保持胚胎湿润。过多的水会导致胚胎浮出对齐，使胚胎难以注入。</li>
	<li>背负荷注射混合到微注射针。使用手绘的玻色酸针填充剂在针头中加入约 0.5 至 1μL 的注射混合物，还可用于凝胶加载管道尖端。注射组合由一个或几个导数RNA（每个80 ng/μL）组成，旨在针对与商业重组的Cas9蛋白（300 ng/μL）混合的特定轨迹。</li>
	<li>从 30 psi 的注射压力开始，按下注射触发器，将空气从针尖排出。这将迫使注射混合到针尖，使注射混合流动。此时，慢慢增加保持/恒定压力，直到注射材料开始流动，然后后退保持/恒定压力，使其略低于从针头流出的注射混合点。门控设置应提供足够的注射材料，以便可以看到少量的材料进入胚胎。 注:在卤烃油下注射沙蝇胚胎，类似于用于 果蝇 和蚊子的其他协议，初步测试，但这些注射的存活率非常低。通过在潮湿的滤纸上切换到注射，如上所述，生存得到了改善。</li>
	<li>将针头插入胚胎侧面。使用胚胎后面的盖片作为后盾，帮助针刺穿胚胎。在轻轻取出针头之前，将少量注射混合物放入胚胎中。</li>
	<li>取出针头后，立即按喷油器从针尖上取出任何回填材料。这将有助于防止针头堵塞。</li>
	<li>进入下一个胚胎。每次注射之间，确保滤纸湿润，针头不堵塞。</li>
	<li>一旦所有胚胎都被注射，计算注射的胚胎数量，以保持运行计数。</li>
	<li>通过在滤纸中加入水来去除盖片，使盖片浮动。此时，使用探针（用粘在尖端的昆虫别针粘住的木材施用器）将滤纸固定到位，同时用手指将盖片从注射的胚胎上拉开。</li>
	<li>一旦盖片被移除，就会从滤纸中清除多余的水。</li>
</ol>4. 注射后饲养 （G0） （图 4）
<ol><li>将滤纸与胚胎一起转移到放置在培养皿的几层湿润滤纸上，以保持湿度。在注射其他胚胎所需的时间将胚胎保留在这里。胚胎最多可保持3小时。注射后，它们会慢慢变成棕色。</li>
	<li>一旦所有胚胎都被注射，手动将它们转移到以前滋润的小尺寸石膏盆上。不要把太多的胚胎放在每个锅里，保持它们之间的距离，以避免个体之间可能的真菌污染。用屏幕覆盖锅，并存储它们，通常会做沙蝇殖民地鸡蛋，保持在孵化器在70%的湿度和26°C，在其整个发展。</li>
	<li>在注射后的第1天，用画笔取出所有受损和死亡的胚胎。在含有注射胚胎的石膏锅中加入 100 μL 的 0.5% 丙酸，一滴一滴，以限制真菌污染。细胞质从注射过程中受损的胚胎中释放，尤其有利于真菌生长。</li>
	<li>每天检查石膏盆，并去除任何坏或死胚胎。如果存在真菌，取出所有受感染的胚胎，并加入几滴0.5%的丙酸。</li>
	<li>第8天和第12天注射后，幼虫孵化。每天两次，将新出现的幼虫转移到新的石膏盆中，最多组5-10个。加入少量食物，每周检查食物2-3次。<ol>
<li>仔细监测幼虫食物的数量，因为过多会大大增加真菌污染的风险。沙蝇幼虫在群体中存活得更好，但是如果没有足够的食物，它们可能是食人族。</li>
	</ol>
</li>
	<li>当幼虫幼崽时，按性别分离它们，以保持雌性为处女。如果有很多幸存者，抛弃雄性，只保留女性。 注:男性和女性都可以携带和传递突变。然而，由于雌性在被穿越之前必须保持处女，因此更容易只使用G0 注射的女性，以避免不得不按性别分离昆虫。G0 男性可以保留作为备份， 如果成年幸存者的数量是低的， 在这种情况下， 将与排序的 wt 处女女性配合。</li>
</ol>5. 突变等位基因的选择和筛选 （图5）
<ol><li>大规模穿越G0 女性与wt男性在笼子里。血液喂养他们完全，将做正常的殖民地维护。</li>
	<li>一天后，用口腔吸气器将每名G0 女性与2-3 wt雄性转移到单独的石膏管中。每天在一个小棉球上用糖喂苍蝇，并使用水注射器，以确保石膏水分适合产卵。</li>
	<li>在G0 雌性产卵（对应于G1）后，将她们的身体收集在单独的、有注释的埃彭多夫管中，供以后的基因化。保持胚胎，通常做wt殖民地。</li>
	<li>通过选择的方法从G0 女性中提取DNA。</li>
	<li>筛选DNA是否存在突变，例如，使用围绕预期CRISPR切割区域的引物设计PCR检测。</li>
	<li>只保留 G1 女性铺设的 G1 胚胎， 以显示转化的证据。当这些G1 胚胎发育成幼崽时，按性别分离它们，只保留雌性。将 G1 女性从同一个 G0 女叉到 WT 男性，用血喂养它们，然后将 2 - 3 名女性和 4 - 5 名男性分成小尺寸容器。 注意:所有G1 个体都有机会携带一个独特的突变，所以如果幸存个体的数量不是太高，那么最好是单独交叉G1 女性与wt男性。几个女性携带不同的突变存在于同一管子的情况下是罕见的，但可能发生。如果在基因化后观察到这种情况，那么G2 后代雌性应该单独与wt雄性配对。这种交配方案避免了后代发生多重突变的可能性。</li>
	<li>产卵后，收集G1 女性身体，并筛选它们的突变。只保留至少有一名女性有突变证据的管子。</li>
	<li>为后代做单对交叉。产卵后，筛选父母存在突变。在每一代人中重复，直到父母双方都是同源的相同突变。一旦确定，它们将成为同源突变种群的创始人。</li>
</ol>

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我们在这里介绍了最近开发的胚胎微注射方法，用于在木薯沙蝇的CRISPR/Cas9靶向诱变。昆虫基因改造的胚胎微注射是在20世纪80年代中期在德罗索菲拉开发的，现在经常用于各种昆虫。其他基因改造材料的交付方法已经开发用于昆虫，如REMOT 20，21，22，23和电波23。然而，胚胎微注射是目前最通用和最有效的交付这些材料的方法。ReMOT作为一种分娩方法非常有前途，应探索用于沙蝇，因为与胚胎微注射相比，成人注射相对容易。然而，到目前为止，ReMot尚未成功用于CRISPR/Cas9 HDR或转子转基因。虽然昆虫胚胎微注射技术与最初开发的技术基本相同，但可能需要对每一个新物种进行技术改进。这些物种差异包括胚胎发育的模式和持续时间、胚胎结构以及胚胎正常发育的环境。沙蝇胚胎特别小（长0.3-0.5毫米，宽0.1-0.15毫米，大约相当于德罗索菲拉卵1的1/3大小）。这种缩小的大小增加了处理鸡蛋而不损坏鸡蛋的难度。由于沙蝇胚胎对干燥特别敏感，因此必须非常仔细地监测协议每一步的水分水平。然而，它们不是水生生物，如果长时间完全淹没在水中，它们就会死亡。嗜血性胚胎微注射需要在胚胎的后端注射。由于沙蝇胚胎的近对称形状，很难确定胚胎的极性。最后，虽然沙蝇胚胎发育过程与果蝇相似，但速度要慢得多。仅胚胎生成就持续6至11天，取决于沙蝇物种，而果蝇卵在产卵后仅孵化一天。嗜血性卵细胞化在卵子沉降后约2小时发生，而沙蝇胚胎在卵子移植后约9小时达到这一阶段。鉴于发育时间的差异，注射针对的是发育中的沙蝇胚胎的中心，假设在发育的早期阶段，同步胚胎中的发育核位于发育胚胎中心附近，从而避免了区分胚胎极性的必要性。
CRISPR/Cas9 在选定的基因组轨迹上创建 dsDNA 中断。这些双链断裂由 NHEJ 或 HDR 在单元格中修复。我们目前采用的方法以前只用于基于NHEJ的突变，它产生小的插入/删除事件，因德尔，导致基因序列的帧移位，导致过早停止结肠和缺乏所有功能域4的蛋白质。昆虫还拥有通过HDR进行dna突破修复的细胞机制，然后可以改道设计更复杂的基因组编辑策略。基于HDR的CRISPR/Cas9基因组编辑仍然需要建立用于沙蝇，并应允许记者的表达和有条件表达突变体的发展，以及其他结构。
这种微注入协议的开发现在打开了使用其他基因组修饰方法的大门，如CRISPR/Cas9 HDR，转子转基因，二元表达系统，如UAS/Gal4，以及phiC31或Cre/lox的现场特定重组。这些其他方法的开发将进一步扩大沙蝇基因表达修饰的工具箱，允许更复杂的基因组操作。然而，在采用这些其他方法之前，需要确定和隔离调节元件，以驱动沙蝇中插入的基因表达，以及插入标记和这些方法的其他组件，如推动转位酶和重组酶表达的促进剂。
最后，非模型昆虫的基因组编辑现在已成为一种可能性，特别是由于CRISPR/Cas9的革命发现和适应15，16。在昆虫中，大多数基因组编辑技术，除了新开发的REMOT技术的显著例外，都需要胚胎微注射，这一步骤既关键又技术要求高。我们希望，本出版物将有助于扩大适应沙蝇的基因表达修饰技术的范围，并导致其生物学的新发现以及莱什马尼亚寄生虫的病媒能力。

病媒传播疾病是持续演变过程中的主要公共卫生威胁。据世界卫生组织统计，数以百计的病媒物种分布在非常独特的植物家族（如蚊子、虱子、跳蚤）中，导致大量微生物病原体的传播，每年导致70多万人死亡。在向量昆虫中，植物素沙蝇（滴虫、心理虫）构成一个庞大的群体，80种经过验证的向量物种表现出不同的表型特征和向量能力，在不同地理区域发现。它们是 列什马尼亚属原生寄生虫的载体，每年造成约130万例新的莱什马尼亚病例和2万至3万例死亡。Leishmanias 的临床结果多种多样，症状从自我限制的皮性病变到内脏传播，在缺乏治疗的情况下是致命的。
沙蝇是严格的陆地昆虫。与其他 Diptera 相比，它们的生命周期相对较长，根据温度、湿度和营养等不同参数，寿命长达三个月。它包括一个胚胎阶段（6至11天），四个幼虫阶段（总共持续23至25天）和一个幼虫阶段（9至10天），然后是变质，然后是成年。沙蝇需要潮湿和温暖的环境来饲养。雄性和雌性都以从花蜜在野外获得的糖为食。只有女性是血液喂养者，因为他们需要从血液中获取蛋白质来生产卵子1。
研究的一个重要重点是确定导致可传播感染发展的病媒/寄生虫相互作用的性质。与其他向量昆虫一样，沙蝇固有的参数已被证明会影响它们的病媒能力，即它们携带病原体和将病原体传染给宿主的能力。例如，木瓜沙飞中古特细胞作为识别寄生虫表面成分的受体表达，可以直接影响其对莱什马尼亚主要2、3的载体能力。昆虫免疫反应途径，免疫缺陷（IMD），也是至关重要的木瓜沙蝇向量能力莱什马尼亚主要4。在伊蚊5、6、7、采采蝇格洛西纳摩尔西坦8和阿诺菲莱斯甘比亚蚊子9、10中，对病媒昆虫免疫反应途径在控制传染性病原体传播方面具有重要作用。
沙蝇/莱什马尼亚相互作用的研究因缺乏适应这些昆虫使用的基因表达修饰方法而受限。直到最近，只有通过小干扰RNA（siRNA）进行的基因下调节进行了11，12，13，14。该技术受成年女性微注射相关死亡率的限制，只导致部分功能丧失，无法代代相传。
CRISPR/Cas9技术彻底改变了沙蝇等非模型生物体的功能基因组研究。CRISPR Cas9系统从原核生物的适应性免疫系统中进行了改造，用于防御噬菌体15、16，并迅速被改造为用于包括昆虫在内的高级真核生物的基因组编辑工具。CRISPR/Cas9 目标基因组编辑的原则基于单一指南 RNA （sgRNA） 与特定基因组的互补性。Cas9核酶与sgRNA结合，在基因组DNA中产生双链DNA（dsDNA）断裂，sgRNA与其互补序列关联。Cas9-sgRNA 复合物由 sgRNA 中的 17 到 20 个互补基座引导到目标序列，然后可以通过两个独立的路径修复 dsDNA 中断:非声端连接 （NHEJ） 或同源定向修复 （HDR）17。NHEJ 修复涉及简单的中断关闭，但经常导致小的插入/删除事件。通过 HDR 修复的 DNA 使用与目标 DNA 共享同源的供体 DNA 分子作为修复模板。昆虫拥有两种机器。
CRISPR/Cas9 技术可以通过 NHEJ 修复途径在选定的轨迹中产生突变;或更复杂的基因组编辑策略，如敲击或表达记者，通过HDR通路与适当的捐赠者模板。在沙蝇中，免疫反应因子的 无 突变等位基因通过NHEJ介导的CRISPR在 木薯4中产生。在另一项研究中还注入了沙蝇胚胎，并结合了针对编码黄色的基因的CRISPR/Cas9组合。尽管如此，没有携带突变的成年人产生18。我们在这里描述了由NHEJ调解的CRISPR/Cas9的沙蝇靶向诱变的详细方法，特别侧重于胚胎微注射，这是协议的关键步骤。

<table><tbody><tr><td>Black Filter Paper 4.25CM PK100</td><td>VWR</td><td>28342-012</td><td>Cut into rectangles that are approximately 46 X 22mm. These are placed between the slide and the coverslip and act as a moist base layer for the embryos during injection.</td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-543A</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Any brand</td><td></td><td>For aligning embryos</td></tr><tr><td>Glass slides</td><td>Fisher Scientific</td><td>12-550-A3</td><td>Base layer of the microinjection set up Figure 2A</td></tr><tr><td>Insect cage</td><td>custom made or several commercial options</td><td></td><td>polycarbonate cage for adults holding and mating Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. &amp;ldquo;Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).&amp;rdquo; Parasite 24. Accessed August 6, 2020. https://doi.org/10.1051/parasite/2017041.</td></tr><tr><td>Larval food</td><td>custom made</td><td></td><td>a mix of rabbit chow and rabbit feces Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. &amp;ldquo;Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).&amp;rdquo; Parasite 24. https://doi.org/10.1051/parasite/2017041.</td></tr><tr><td>Microcaps 100 ml</td><td>Drummond</td><td>1-000-1000</td><td>Used to back fill microinjection needles</td></tr><tr><td>Mouth aspirator</td><td>John W. Hock Company</td><td>Model 612</td><td>mouth aspirator with HEPA filter</td></tr><tr><td>Olympus SZX12</td><td>Olympus Life Sciences</td><td></td><td>Microinjection microscope</td></tr><tr><td>Ovipots</td><td>Nalge company</td><td></td><td>ovipots are made from 125-ml or 500-ml straigh-sided plolypropylene jars modified by drilling 2.5cm holes in the bottom and filled with 1cm of plaster of Paris. Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. &amp;ldquo;Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).&amp;rdquo; Parasite 24. Accessed August 6, 2020. https://doi.org/10.1051/parasite/2017041.</td></tr><tr><td>Paint Brush 6-0</td><td>Any Art Supply Company</td><td>n/a</td><td>Used for aligning embryos</td></tr><tr><td>Propionic acid</td><td>Sigma-Aldrich</td><td>402907</td><td>antifungal agent</td></tr><tr><td>Standard Glass Capillaries</td><td>World Precision Instruments</td><td>1B100-3</td><td>Used for making microinjection needles</td></tr><tr><td>Trio-MPC100 Controller and MP845 Manipulator</td><td>Sutter Instruments</td><td></td><td>Microinjection Controller and Micromanipulator</td></tr></tbody></table>

sand fly (phlebotomus papatasi) embryo microinjection for crispr/cas9 mutagenesis

沙蝇是 莱什马尼亚 物种的自然载体，原生动物寄生虫产生从皮性病变到内脏病理学等多种症状。破译向量/寄生虫相互作用的性质对于更好地了解 莱什马尼亚 传染给宿主至关重要。在控制沙蝇向量能力的参数（即它们携带和传播病原体的能力）中，这些昆虫固有的参数被证明发挥着关键作用。例如，昆虫免疫反应会影响沙蝇向量对 莱什马尼亚的能力。由于缺乏适合用于这些非模型生物体的基因表达修饰方法，对此类参数的研究受到限制。通过小干扰RNA（siRNA）来降低基因调控是可能的，但除了在技术上具有挑战性之外，沉默只导致部分功能丧失，无法代代相传。CRISPR/Cas9技术的靶向突变最近被改编成 木薯沙 蝇。这项技术导致在一个专门选择的轨迹中产生可传播的突变，从而能够研究感兴趣的基因。CRISPR/Cas9 系统依赖于定向双链脱氧核糖核酸断裂的诱导，后来通过非同源端连接 （NHEJ） 或同源驱动修复 （HDR） 进行修复。NHEJ 包括一个简单的休息关闭，并经常导致小插入/删除事件。相比之下，HDR 使用与目标 DNA 共享同源的供体 DNA 分子的存在作为修复模板。在这里，我们提出了一种沙蝇胚胎微注射方法，用于CRISPR/Cas9使用NHEJ靶向突变，这是迄今为止唯一适应沙蝇载体的基因组修饰技术。

CRISPR/Cas9微注射协议在这里描述产生沙蝇突变体是在以前的出版物4中建立的。这种方法产生了高效的诱变，因为540个人中有11人幸存下来，其中9人是突变者。在为CRISPR/Cas9突变设计指南时，关键的第一步是对周围区域进行排序，以便成为目标。测序模板应该来自将用作注射胚胎来源的菌株。仅仅依靠已公布的基因组序列来设计指南是有风险的。在已发布的基因组和指南前设计序列之间存在差异并不罕见。在某些情况下，当目标基因周围的区域被测序时，不存在由已公布的基因组序列设计的指南（Harrell，个人观察）。此外，该序列可能包括单核苷酸多态性 （SNPs），在基因化过程中可能被误认为是真正的 CRISPR 编辑。因此，确认目标区域的顺序对于协议的其余部分取得成功至关重要。
通过微注射对昆虫进行成功的基因改造主要取决于两个关键方面:在发育的适当时间交付材料（蛋白质、质粒或mRNA），对胚胎的损害尽可能小:饲养健壮、健康的昆虫，使这些昆虫在手术中存活下来并产生后代。这个程序的初始阶段，在 图2中描述，从从wt菌落的雌性血液喂养开始，并在五天后进入胚胎采集和微注射。第二阶段包括饲养注射的胚胎直到成年，使适当的交叉，并识别和隔离感兴趣的突变。
用于注射的胚胎收集30-60分钟，以便确定输卵后数小时的相对年龄。然后允许胚胎在注射开始前发育3小时。这个昆虫发育的窗口允许胚胎在注射中存活下来。在此老化期后，胚胎被收集到准备的注射滑梯上，胚胎用细刷在盖片边缘滚动到位。最终配置显示在图 3A 中。重要的是要湿过滤纸足够，使半月板形成在盖滑边缘的胚胎坐。太多的水和胚胎将被推离盖滑边缘。太少会导致胚胎被拉到盖子下。胚胎需要保持湿润，否则胚胎膜变得难以注入。盖片边缘充当后挡，使针头在压在边缘时穿透胚胎。它是锋利的针头和后挡的组合，使针头能够顺利穿透。
与大多数成功的微注射方案一样，适合注射胚胎的良好微注射针也很重要。好的，锋利的针头被定义为容易穿透胚胎的针头，而不允许材料在注射后逸出。当针头滑入胚胎时，良好的渗透是显而易见的，在渗透过程中很少或根本没有凹痕胚胎膜，针头取出后胚胎也没有物质泄漏。良好的锋利的针头是使用针拉器设置，产生一个针，来到一个细点（图1A）。拉针不应有太长的锥度。否则，针头的流明在锥度的大部分（图1B）中变得非常狭窄，并且很难获得足够高的注射压力，迫使材料穿过针头。在此协议中，使用了斜线针头。拉针和斜线的过程在梅蒂和哈雷尔19中描述。在沙蝇胚胎长期发育的情况下，有锋利的针头，尽量减少对胚胎的损害，从而防止材料在注射部位泄漏尤为重要。当胚胎质在注射后从胚胎中泄漏时，这种材料是霉菌和真菌生长的丰富媒介。在较短时间内发育的胚胎中，胚胎可以在模具成为问题之前发育和孵化。注射后明显泄漏胚胎的胚胎应切除。
在注射过程中，必须根据需要加入水，将细画笔润湿并触摸到滤纸中，必要时重复，直到水的半月板刚刚在胚胎底部。需要注意注射当天的相对湿度。在低湿度的日子里，需要更多的水。注射完成后，在滤纸上加入少量额外的水，使盖片稍微浮动，从而更容易去除。一旦盖片被移除，注入胚胎的滤纸就会被弄脏，使滤纸几乎不潮湿。然后，胚胎可以转移到湿润的石膏锅中，用精美的画笔孵化。在现阶段，胚胎非常脆弱，因此必须非常小心地进行这一过程。防止胚胎相互接触也很重要，以便能够去除发霉的胚胎并限制真菌污染的传播。
注射后，G0 注射的胚胎按正常饲养程序保存在石膏盆上。在他们孵化之前，注射的胚胎盆应该每天检查一次，以去除不健康的胚胎。 图4A 介绍了从产卵和注射到成年的G0 个体饲养的预期时间表。 图4B 显示了G0 胚胎健康且应保留的例子:或损坏、被真菌污染、干燥或变形，应丢弃。G0 注射的个体被认为是突变等位基因的马赛克。应设计一种识别潜在突变等位基因的方法，例如对预期切割地点周围的区域进行 PCR 分析。 图 4C 展示了一个 PCR-分析示例，显示马赛克 G0 个体，显示与预期 wt 产品额外的 PCR 产品，表示突变等位基因。
一旦成人出现，从 G0 注射胚胎发育的 G0 雌性与 wt 雄叉，允许产卵，并在以后被基因化。只保留含有 G0 苍蝇的管子，显示选择的基因型方法突变的证据。下一代（ G1 雌性）的苍蝇要么与雄叉，要么在兄弟姐妹之间单独交叉（来自G2 ）。这些十字架被允许产卵，然后通过选择的方法基因。最后一步重复，直到获得同源突变的男性和女性，建立一个同源突变线。 图5A中给出了适当继承十字架以建立突变线的示意图表示。 图 5B 显示了一个基因型 PCR 允许识别同源突变兄弟姐妹十字架的示例。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61924/61924fig01.jpg"> 图1:微注射针。好的针头。B. 针与极端锥度不适合注射。 <a href="https://www.jove.com/files/ftp_upload/61924/61924fig01large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61924/61924fig02.jpg"> 图2:胚胎采集和微操纵概述（此图改编自4）。<a href="https://www.jove.com/files/ftp_upload/61924/61924fig02large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61924/61924fig03.jpg"> 图3:微投影设置。A. 微注入设置的示意图表示。B. 微注射对齐胚胎的特写。 <a href="https://www.jove.com/files/ftp_upload/61924/61924fig03large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61924/61924fig04.jpg"> 图4:马赛克G0 成年个体的饲养和鉴定。A. 从胚胎微注射到 G0 成年的预期时间安排。B. 注射后好看和不好看的胚胎的例子。C. 一个经过改造的 G0 马赛克个体的 PCR 基因化。 <a href="https://www.jove.com/files/ftp_upload/61924/61924fig04large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61924/61924fig05.jpg"> 图5:识别和隔离感兴趣的突变。A. 分离突变等位基因和建立同源突变种群的实验策略的原理图表示（ 图4）。颜色表示携带突变等位基因的细胞在异质（粉红色）或同源（红色）状态下的存在。B. 用于基因化单个沙蝇交叉的 PCR 筛选策略示例。 <a href="https://www.jove.com/files/ftp_upload/61924/61924fig05large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>

Watch this Scientific Journal Video about Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis at JoVE.com

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

该协议详细介绍了CRISPR/Cas9在沙蝇中靶向诱变的步骤:胚胎采集、注射、昆虫饲养和识别，以及兴趣突变的选择。

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

sand-fly-phlebotomus-papatasi-embryo-microinjection-for-crisprcas9

Nanyang Technological University

Research

JoVE Journal

Genetics

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

7.1K 次观看.  National Institutes of Health. 该协议详细介绍了CRISPR/Cas9在沙蝇中靶向诱变的步骤:胚胎采集、注射、昆虫饲养和识别，以及兴趣突变的选择。

视频: Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

果蝇中 phiC31 整合酶介导的胚胎转基因

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

作者聚焦:优化胚胎显微注射以促进果蝇的转基因

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

胚胎显微注射用于果蝇转基因

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

行为学

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

摘要

探索更多视频

此视频中的章节

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

CRISPR/Cas9基因组编辑的半球藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

摘要

探索更多视频

此视频中的章节

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

CRISPR/Cas9基因组编辑的半球藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎