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 &#181;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 &#176;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>
		</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

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

临界条件在免疫染色豌豆蚜虫胚胎鉴定:增加组织通透性和降低背景染色

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

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 ...

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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