TO 和 TD 分别得到 JSPS KAKENHI 赠款编号 19H02970 和 20H02999 的支持。

1. 实验室菌落的维护
<ol><li>为了维持野生类型和突变种群，使用大型塑料容器（460 毫米 x 360 mm x 170 mm）与常规人工鱼食、顶部有通风孔的塑料杯中的水、用于隐藏昆虫的折叠纸和用于产卵的分层棉花（图 2A）。将所有 T. 家用 文化保留在 37 °C 孵化器内，并将每个容器内的相对湿度 （RH） 设置为 60%-80%。 注:由于 T.家政 从大气中吸收水蒸气11，因此不需要直接供水。适当的 RH 是由于每个容器内装有通风孔的塑料杯或无盖杯中存在水蒸气而保持的。无需在包含培养物的整个孵化器内加湿。在此协议中描述的条件下，每个发育阶段的大致持续时间显示在 图 1中。发展速度可以通过改变温度和/或RH12来调整。</li>
	<li>定期添加食物。在水干涸之前加水。</li>
	<li>鉴于成年人停止在肮脏的环境中产卵和/或人口稠密，至少每3个月将人口转移到一个新的清洁容器（见 讨论）。</li>
</ol>2. 鸡蛋收集和微弹射
<ol><li>设计指南RNA（gRNA）<ol>
<li>为每个所需目标设计一个 gRNA 序列，并将 gRNA 序列与基因组组装对比，以检查可能的脱靶识别站点。</li>
		<li>根据制造商的说明8合成和净化 gRNA。 注:例如，在针对ATP绑定盒式运输机 白色 基因的情况下，设计了20 bp目标序列，并订购了两个合成的DNA寡核苷酸5'-TAATACTACTATAGTAGTAGGTGGAC-3'和5'-TTC-塔卡特卡塔-3'。DNA模板是通过对这两种寡核苷酸进行退化，然后进行PCR放大，然后用T7 RNA聚合酶从模板中体外转录。</li>
	</ol></li>
	<li>准备鸡蛋收集殖民地<ol>
<li>将大约20名男性和20名女性成人转移到一个中等大小的容器（200毫米×150毫米×90毫米），内有食物、水供应、一张折叠纸和一小块分层棉，用于产卵（图2B）。</li>
		<li>建立几个殖民地，在短时间内获得大量阶段性胚胎，用于基因组编辑。 注:大约20-40个鸡蛋预计将在37°C的8小时后从殖民地收集。 转移的成年人通常需要几天时间才能开始产卵，这可能是由于适应了新的环境。</li>
	</ol></li>
	<li>在注射当天，用新的代替容器内的棉花。</li>
	<li>将 76 毫米 x 5 毫米双面胶带放在常规 76 mm x 26 mm 玻璃滑梯上。</li>
	<li>8小时后，使用钳子将层分离，从分层棉中收集鸡蛋。</li>
	<li>使用湿漆刷将双面胶带上的鸡蛋对齐，并在鸡蛋之间保持 2 mm 的距离。所有鸡蛋应定向，使鸡蛋的纵轴面对注射侧（图3A）。在注射过程中，用油漆刷轻轻按压鸡蛋，以牢固地握住鸡蛋。 注:在此协议中，真菌在潮湿和温暖的条件下在受损的注射卵子中快速生长。重要的是保持鸡蛋之间的距离，以防止交叉污染和真菌的扩张。</li>
	<li>将 gRNA 和 Cas9 蛋白质分别混合到 100 ng/μL 和 500 ng/μL 的最终浓度。使用蒸馏水进行稀释。在室温下孵化混合物10分钟，促进核糖核蛋白复合形成，然后保持混合在冰上。 注:在1%的最终浓度下，中性红可以添加到注射溶液中，以监测注射量。</li>
	<li>将 gRNA/Cas9 溶液的 2 μL 装载在带微型装载机的玻璃注塑毛细管中。在注射前确保溶液中没有气泡。如有必要，点击针头去除气泡。 注:在这个实验中，一个现成的针头被用来获得一个细针尖（图3B）。一种形状相似的自制针头可以代替。</li>
	<li>将以前装有 gRNA/Cas9 溶液的玻璃喷射毛细管固定在装有操纵器的支架上，并将支架连接到电子微注射器。</li>
	<li>优化针尖的形状，用钳子稍微打破它，以防止堵塞和获得更好的耐久性在整个一系列注射（一个适当的针的例子显示在图3C）。 注意:建议在针头堵塞时更换针头。如果钳子再次断裂，可以继续注射同一针，但更宽的尖端会导致更多的卵子损伤，降低其存活率。由于 T.家蛋 柔软易碎，保持细针尖是注射后存活率高的关键。</li>
	<li>将针头插入鸡蛋纵向轴的中点，并注射少量溶液（见 图3D-H， 以参考注射量）。根据针尖的形状，在注射过程中调整电子微注射器的配置。 注意:建议在注射过程中施加恒定压力，否则粘性鸡蛋含量很容易流入玻璃针，并可能堵塞。解决方案可能是注射短压力脉冲或恒定压力，具体取决于注入的液体量。请记住，当针尖有一个宽开口，太多的溶液被注射到鸡蛋，这会导致致命性（图3F-H）。在这种情况下，通过恒定的压力保持恒定的液体流动，然后将针头插入鸡蛋中并立即将其拉出。如果它导致太多的溢出或鸡蛋爆裂，改变针。</li>
	<li>将注射的鸡蛋放在一个容器中，其大小与注射鸡蛋的数量相当（+lt;20鸡蛋:小菜;+20个鸡蛋:中型容器），60%-80%RH和37°C。</li>
</ol>3. 交配
<ol><li>定期检查注射的鸡蛋，用钳子丢弃受损的鸡蛋，以避免真菌生长（图3I，J）。万一鸡蛋上长出太多的真菌，用 70% 的 EtOH 清理鸡蛋表面。</li>
	<li>孵化前（产蛋后约10天，37°C），将玻璃滑梯与注射的鸡蛋浸入滑石粉中，涂上双面胶带的表面。这将避免孵化的仙女堆叠。将粉末涂层的玻璃滑梯转移到一个中等大小的容器中，内有食物、水和折叠纸，用于隐藏昆虫。</li>
	<li>在仙女孵化后取下玻璃滑梯。定期供应食物，直到他们成年。 注:个体孵化后大约需要2.0-2.5个月才能成年（图1A）。成人阶段的判断依据是女性发育良好的卵母（图1B）。</li>
	<li>要与个体交配，请将尽可能多的野生型女性成人从实验室殖民地转移到中型容器，并在 37 °C 下孵育至少 14 天，以确保她们是处女。 注:没有必要从实验室殖民地收集处女，因为成年T.家庭有一个反复的循环的熔融和交配称为"生殖和融色周期"，在此期间，女性扔掉精子与每个摩尔和伴侣再次在下一个受精周期13。</li>
	<li>将从注射卵子和野性成人发育成小塑料盘（+100 mm x 40 mm）的男性或女性G0成人转移到带食物、折叠纸和一小块棉花的塑料盘上，用于产下G1卵子（图2C';交配盘）。将配菜放在一个更大的容器中，带 60%-80% RH（图 2C）。 注:多个野生类型的成人可以包括在一个菜，以增加成功交配的机会，虽然高成功率已经实现了一对一的配对。</li>
</ol>4. 基因型
<ol><li>为每个 gRNA 设计 PCR 入门对，以放大 100-200 bp 产品，其中包括 gRNA 所针对的站点。对基因组组装的每个引体序列进行爆炸，以检查其特异性（图 4A中针对白色基因的示例示例）。</li>
	<li>检查 G0 成人的细菌线转换。<ol>
<li>卵子产卵五天后，从每个G0成人对收集单独的G1卵子，放入0.2 mL管中（每管1个鸡蛋;将采集的样品储存在-20°C，用于长期储存）。分离棉层收集鸡蛋。 注:建议从每对交配对中至少产生12个G1仙女，以评估生殖系传播的成功率（见 代表结果）。</li>
		<li>在每个管子中加入 0.25 毫克/mL 蛋白酶 K 溶液（溶解在 Tris-EDTA 缓冲区）的 15 μL，用牙签短暂均质样品，并在 55 °C 下孵育 3 至 16 小时。</li>
		<li>将样品放在95°C下10分钟，使蛋白酶K失活。</li>
		<li>将 90 μL 蒸馏水加入每个管子中，并混合好。在包含第 4.1 步设计的引漆的 10 μL PCR 反应组合中使用 2 μL 的超常剂。 注:建议使用针对粗模板优化的DNA聚合酶，以达到足够的PCR放大。</li>
		<li>要分析 PCR 产品，请使用微芯片电泳系统执行异质多式移动检测 （HMA）（图 3B;参见 Ohde 等人，2018 年）8。 注:突变可以用两种替代方法进行评估:（1）HMA与标准凝胶聚合物，如8%聚丙烯酰胺14:（2）用T7内核酶消化PCR产品，然后消化凝胶电磷酸酶15。</li>
		<li>只保留G1仙女产生的G0成人，其中含有突变的细菌线，并丢弃其他。</li>
	</ol></li>
	<li>G1仙女/成人的个体基因成型<ol>
<li>用吸气器或画笔将G1仙女隔离成24口井板。将 24 井板放置在较大的容器中（例如本协议中使用的中型容器），并配上上述水源，以保持 60%-80% 的 RH（图1D）。保持人工常规鱼食的供应（图1D'）。 注:虽然此步骤可以在仙女和成人阶段的任何时间点执行，但建议在成年后和配对前执行此步骤（+注射后 2.5 个月; 图1B）因为它更容易在一个大容器中维护火炉。需要单独饲养，以跟踪以下步骤上每个G1仙女的基因型。来自同一G0成人的G1仙女可以有不同的突变。</li>
		<li>用钳子从仙女/成人那里捏取和拉 ceci 和 caudal 灯丝，并将它们收集到含有 50 μL EtOH 的 0.2 mL 管中（将收集的样品存放在 -20 °C 处，以进行长期存储）。 注:组织样本在EtOH中收集，因为它可以防止这些小样本因静电而丢失。如果需要停止昆虫的运动，在采集组织样本时在冰上麻醉仙女/成人。由于 T.家药 在冰上长期冷却后无法存活，因此不要麻醉超过一分钟，并立即将它们移回室温。子宫颈和颅丝的消融不会增加死亡率。</li>
		<li>将盖子打开的样品管放在热块上 15 分钟，在 70 °C 下蒸发 EtOH。</li>
		<li>重复步骤 4.2.2-4.2.5 用于基因成型。</li>
		<li>将观察突变波段模式的 PCR 产品提交到标准桑格测序服务中。</li>
		<li>保持G1仙女/成人与所需的突变，并丢弃其他（见 图4C 的代表性测序结果）。</li>
	</ol></li>
	<li>穿过含有配菜中所需突变的成人，获得下一代，以建立同源突变菌株。</li>
</ol>

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作者没有什么可透露的。

对于成功生成所需的 T.家 用突变体CRISPR/Cas9，首先重要的是收集足够数量的分阶段胚胎进行注射。对于足够数量的 T.家蛋 的不断收集，关键是选择一个适当的大小的容器，以具有较低的人口密度，因为它将有助于成功完成一系列复杂的交配行为，这是重复后，每个成人摩尔13。男性 T.家庭 成人通过精子间接地将其精子转移给女性。斯威特曼（1938年）报告说?...

热比娅家居属于最基础的昆虫之一，Zygentoma，它保留了祖先的代谢和无翼生命周期。这种基底植物遗传位置和祖先特征为研究地球上昆虫成功背后的机制树立了有吸引力的模型，昆虫覆盖了描述的动物物种1的70%以上。长期以来，由于其作为实验室模型的合适特征，如从胚胎到生殖成人的寿命相对较短（2.5-3.0个月），因此主要用于研究昆虫生理的祖先特征:图1A）和一个简单的繁殖。在过去的三十年里，它的使用已经扩大，以研究各种特征的祖先特征，如身体计划，神经分化，昼夜节律2，3，4。
先进的遗传工具在T.家政的应用可以进一步加速这种贡献，在广泛的研究领域。成功的RNA干扰（RNAi）介制的基因敲击在胚胎，仙女和成人已经报告在T.家庭4，5，6。系统性RNAi的效率仍然高度依赖物种-例如，它通常是高在科洛普泰拉，而它是低在麻风病顺序7。T. 家政的 RNAi 击倒的效率和持续时间尚未评估。除了RNAi，我们之前已经报告了成功的CRISPR/Cas9介质基因敲除在T.国内a8。CRISPR/Cas系统已广泛应用于昆虫的基因组编辑，特别是针对基因敲除。在建立CRISPR/Cas系统组件进入核9的协议后，通过敲击外源构造，可以扩大其用途，用于其他应用，如基因报告器检测、细胞谱系跟踪和转录活性操纵。结合已公布的基因组组装10，在T.家谱中广泛使用和进一步发展CRISPR/Cas为基础的基因组编辑将有助于研究昆虫卓越适应性成功背后的进化机制。在这里，我们描述了胚胎微射和交配成人T.家用，以产生突变株使用CRISPR/Cas9的详细协议。考虑到这种新方法，我们讨论了考虑非传统模型物种的独特生物学对于这些技术的成功应用的重要性。

<table><tbody><tr><td>24-well plate</td><td>Corning</td><td>83-3738</td><td></td></tr><tr><td>Alt-R S.p. HiFi Cas9 Nuclease V3, 100 &amp;micro;g</td><td>Integrated DNA Technologies</td><td>1081060</td><td></td></tr><tr><td>Anti-static cleaner</td><td>Hozan</td><td>Z-292</td><td>for removing static electricity from a 24-well plate</td></tr><tr><td>Barrier Box 20.7L</td><td>AS ONE</td><td>4-5606-01</td><td>Large container</td></tr><tr><td>FemtoJet 4i</td><td>Eppendorf</td><td>5252000013</td><td>Electronic microinjector</td></tr><tr><td>Femtotip II, injection capillary</td><td>Eppendorf</td><td>5242957000</td><td>Glass injection capillary</td></tr><tr><td>High Pack 2440mL</td><td>AS ONE</td><td>5-068-25</td><td>Middle-sized container</td></tr><tr><td>Incubator</td><td>Panasonic</td><td>MIR-554-PJ</td><td>for 37 &amp;deg;C incubation. No need to humidify inside the incubator.</td></tr><tr><td>KOD Fx Neo</td><td>Toyobo</td><td>KFX-101</td><td>PCR enzyme for genotyping. Optimized for an amplification from crude templates.</td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-8</td><td>for holding the micromanipulator</td></tr><tr><td>Microloader</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MM-3</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>SZX12</td><td>for microinjection. More than 35X magnification is sufficient for the microinjection</td></tr><tr><td>MultiNA</td><td>Shimadzu</td><td>MCE-202</td><td>Microchip electrophoresis system</td></tr><tr><td>NiceTac</td><td>Nichiban</td><td>NW-5</td><td>Double-sided tape to place eggs on a glass slide</td></tr><tr><td>Paint brush (horse hair)</td><td>Pentel</td><td>ZBS1-0</td><td></td></tr><tr><td>Plant culture dish</td><td>SPL Life Sciences</td><td>310100</td><td>Mating dish and water supplies for a large and middle-sized containers</td></tr><tr><td>Proteinase K, recombinant, PCR Grade Lyophilizate from Pichia pastoris</td><td>Roche</td><td>3115836001</td><td></td></tr><tr><td>SZX 12 microscope</td><td>Olympus</td><td>SZX 12</td><td>More than 35X magnification is sufficient for the microinjection</td></tr><tr><td>Talcum powder</td><td>Maruishi</td><td>877113</td><td></td></tr><tr><td>Tetra Goldfish Gold Growth</td><td>Spectrum Brands</td><td></td><td>Artificial regular fish food</td></tr></tbody></table>

egg microinjection and efficient mating for genome editing in the firebrat thermobia domestica

火布拉特 热比亚家是 一种无翼的无翼物种，适合研究昆虫的发展机制，导致它们在地球上成功的进化辐射。基因工具（如基因组编辑）的应用是理解基因变化的关键，这些变化是 Evo-Devo 方法中负责进化过渡的关键。在这篇文章中，我们描述了我们目前的协议，以产生和维持 T.国内突变菌株。我们报告一种干注射方法，作为报告的湿注射方法的替代品，使我们能够获得注射胚胎的稳定高存活率。我们还报告了一个优化的环境设置，以配合成人，并获得后代的高效率。我们的方法强调了将每个物种独特的生物学考虑在非传统模型生物中成功应用基因组编辑方法的重要性。我们预测，这些基因组编辑协议将有助于实施 T.家用作为 实验室模型，并进一步加快该物种中有用的遗传工具的开发和应用。

在我们手中，大约100个鸡蛋可以很好地注射一个单一的注射毛细亚时，它有足够的尖端（图3C）。在产卵后的前8小时内在胚胎中注射gRNA/Cas9核糖核蛋白复合物，导致在gRNA靶点产生因德尔。这会导致注射一代 （G0） 的某些细胞发生胆汁突变，因此在 G0 中通常获得突变马赛克表型。例如，当本协议用于注射旨在瞄准 白色 基因的 gRNA 时，32.6% 的 G0 仙女在其复合眼睛和背?...

Watch this Scientific Journal Video about Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica at JoVE.com

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

我们为卵子的饲养、微喷射和在基因组编辑后生成和维持突变菌株的火布拉特 热比亚家用菌 株的有效交配提供了详细的协议。

卵子微弹射和有效交配的基因组编辑在火布拉特 热比亚家

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

egg-microinjection-efficient-mating-for-genome-editing-firebrat

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

4.3K 次观看.  Kyoto University. 我们为卵子的饲养、微喷射和在基因组编辑后生成和维持突变菌株的火布拉特 热比亚家用菌 株的有效交配提供了详细的协议。

视频: Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

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 ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

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, ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

注射格里鲁斯双形蛋

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

发育生物学

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 Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

生物工程

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.

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

生物学

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

卵子微弹射和有效交配的基因组编辑在火布拉特热比亚家

摘要

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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

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

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

注射格里鲁斯双形蛋

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

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化

卵子微弹射和有效交配的基因组编辑在火布拉特 热比亚家

摘要

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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

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

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

注射格里鲁斯双形蛋

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

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化

卵子微弹射和有效交配的基因组编辑在火布拉特热比亚家

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