这项工作得到了加利福尼亚的一所慷慨的大学, 河滨 (UCR) 实验室启动基金的支持, 操作系统 a, 美国农业部国家食品和农业研究所 (NIFA) 孵化项目 (1009509) O.S.A。

1. n 金小蜂菌落饲养
<ol>
	<li>设置几个殖民地 (〜 3-5) 通过放置 200-500 N. 金小蜂成人 (与3:1 的女性: 男性的比例) 在臭虫宿舍笼子。 注意: 除了使用臭虫宿舍以外, 黄蜂还可以在多个小玻璃试管中传播, 用棉花塞上大约15-20 黄蜂/瓶。<ol>
		<li>将黄蜂保持在25±1° c, 30% 相对湿度和12:12 光暗循环, 每天用小滴 1:10 (v/v) 蔗糖/水溶液喂养它们。 注: 黄蜂的繁殖和收集也可以在室温下进行, 不受控制的湿度;然而, 低于25° c 的温度会略微降低胚胎的发育时间。</li>
	</ol>
	</li>
	<li>允许黄蜂交配至少4天前注射。<ol>
		<li>在头两天, 允许交配雌性饲料新鲜的bullata蛹, 以及小滴 1:10 (v/v) 蔗糖/水溶液。</li>
		<li>在接下来的两天里, 去掉苍蝇蛹, 以剥夺雌性黄蜂的产卵场所, 使其非常妊娠。</li>
	</ol>
	</li>
</ol>
2. n 金小蜂前胚阶段胚胎的收集和排列
<ol>
	<li>允许雌性黄蜂寄生 (排卵胚胎) 进入年轻的寄主 (S. bullata蛹)。为了使主机保持新鲜, 在获得这些主机后立即将其存储在4° c 中, 并在需要时才删除。 注意: 年轻的主机可以由红色的蛹, 而较旧的主机有一个较深的彩色蛹 (图 1A)。<ol>
		<li>将单个新鲜的bullata蛹放入一个具有 pupae-sized 孔切入中心的泡沫塞。只暴露〜0.2 厘米的前端 (首选产卵点) 的主机, 以最大浓度的寄生和更容易的胚胎收集。 注意: 前端是圆的, 而后端较粗, 并包含一个 "火山口状" 开口 (图 1B)。</li>
		<li>将寄主蛹 (大约每100只黄蜂的5寄主蛹) 放入笼中, 等待大约45分钟, 让黄蜂寄生它们 (图 2- ii)。 注意: 非常重要的是, 胚胎是尽可能年轻的, 最好是在第一个小时的 oviposited, 以确保他们在 pre-blastoderm 阶段。不应注射陈旧的胚胎 (#62; 1.5 小时)。</li>
		<li>通过手工检索和轻轻刷/吹掉任何居住的黄蜂, 删除寄生主机。每〜15分钟后, 更换新的主机, 以继续收集在注射期间早期阶段的鸡蛋。</li>
	</ol>
	</li>
	<li>用手从笼子里取出刚寄生的主机。在解剖显微镜下, 使用镊子, 仔细剥离外部蛹 (蛹壳) 的后端 (0.4 厘米), 以揭露N. 金小蜂卵 (图 2- iii)。 注意: 必须小心避免穿刺蛹皮肤;如果发生这种情况, 血淋巴将滋润黄蜂的胚胎, 这将是非常难以去除的微注射。</li>
	<li>使用液体粘合剂将片粘附在干净的玻片上, 以准备胚胎对齐幻灯片 (图 3)。 注: 一种高性能的瞬时粘合剂用于提高粘结强度和干燥速度, 但是任何能使片在滑动中移动的胶水都是可以接受的。</li>
	<li>小心地用湿细尖的画笔将胚胎从寄主中拒绝, 确保不损害寄主的软蛹皮肤。 注: "胚镐", 它实质上是一对超细镊子的一半, 可代替画笔使用。</li>
	<li>转移〜20胚胎一个一个到幻灯片, 立即毗邻的片与湿画笔。将胚胎的前端朝向盖子滑动的边缘方向, 以便胚胎的后端指向相同 (图 2- iv)。这将使得在所有胚胎中注入相同位置的精确度更高。 注: 此改进后的技术不需要双面粘胶带来修复与先前2122所描述的幻灯片有关的胚胎。此外, 不要枯萎胚胎, 或在注射过程中用烃油覆盖鸡蛋, 如前所述, N. 金小蜂胚微注射22。</li>
</ol>
3. Microinjections 针的制备
<ol>
	<li>将硅酸铝毛细管玻璃放入针拔器中。 注: 良好的针对于成功的金小蜂胚胎注射至关重要。注射针应该有一个尖锐的和细微的尖端, 以便通过绒毛膜容易穿透。还可以使用石英和硼硅酸盐毛细管 (图 4)。</li>
	<li>将热设置为 605,速度到 130,延迟到 80,拉到 70, 以及压力至500针拔器。 注: 在表 1中可以看到用于拉动石英和硼硅酸盐针的额外的拔针器设置;不同的车夫和细丝的参数可能有所不同。</li>
	<li>激活拔针器以创建正确的针。根据需要重复生产多个针头。 注: 拉针可以储存在一个培养皿中, 其中包含多个平行卷的商业建模者的粘土。</li>
	<li>在25° c (图 2- iii) 上, 在十年代左右的时候, 将针尖与金刚石研磨板略微接触。 注: 注射针头通过促进进入胚胎, 减少损伤, 同时提高了通过针流的董事, 从坡口受益。</li>
</ol>
4. 胚显微注射
<ol>
	<li>准备由基因组修饰试剂组成的注射混合物 (例如,转 + 帮助剂转, 或 CRIPSR 试剂,等.), 并把它放在冰上。</li>
	<li>使用 microloader 贴士, 用2µL 的注射剂装入注射针。 注: 该协议中所示的注入混合物包括 single-guide RNA (sgRNA) 和重组 Cas9 蛋白混合在 H2O 中。</li>
	<li>将带有内衬胚的玻璃玻片放在复合显微镜的舞台上。</li>
	<li>小心地将针插入胚胎的后端, 垂直角度为25-35 °。</li></ol>注入 1-5 pL 注射混合物 (约2-10% 的胚胎的体积), 并确保胚胎似乎膨胀略有混合物注入到它 (图 2- iv)。 注: 如果在胚胎中注入过多的注射混合物, 它可能会从胚胎中迸发出细胞质。此外, 不适当的斜面或折断的针头太大的开放也可能导致胚胎破裂。<ol>
		<li>如果发生堵塞, 尝试 re-beveling 针。金小蜂胚的细胞质异常黏稠, 粘稠, 导致频繁的针头堵塞 (1/25 注射)。 注意: 重要的是要保持在注射期间的胚胎湿润, 定期添加去水使用画笔。在刷子上的水的数量是关键的移动胚胎周围和协调与轻松。过多的水导致胚胎漂浮, 过少的水使它们很难四处走动。</li>
		<li>一次注射〜20-40 鸡蛋 (应采取约10分钟), 然后停止。通过轻轻地接触注射过的鸡蛋并将它们放入寄主中, 将注入的鸡蛋与湿的画笔一起转移。然后继续使用新鲜的新产卵的一批鸡蛋 (和 #62, 1 岁) 再次注射。 注意: 理想情况下, 如果一个人不断地收集和排列鸡蛋, 而另一个人在注射基因组修饰成分和移植胚胎到寄主蛹, 这个协议是最有效的。</li>
	</ol>
	

5. 将G0 金小蜂胚胎移植到前蛰寄主上
<ol>
	<li>小心地将注入的 G0胚胎放在先前被蜇过的bullata蛹 (图 2- vi) 上, 并在注射后用湿的画笔。 注: 用于收集用于显微注射的胚胎的寄主蛹将为此目的而工作。此外, 尽管有很大的努力, 目前还没有可用于22的人工饮食。<ol>
		<li>在一个单一的 pre-stung 寄主上放置多达40次注射的胚胎, 以避免过度拥挤和幼虫的竞争。 注: 注射会对胚胎造成损害;不小心移植胚胎的寄主蛹可能挤压注射成分或蛋黄, 并导致死亡的胚胎。</li>
	</ol>
	</li>
	<li>将寄主蛹放入带有湿滤纸和棉球的培养皿中, 在25° c 下孵育, 大约70% 的湿度, 直到注入胚胎孵化 (大约1-2 天) (图 2- vii)。 注意: 重要的是, 主机可以留有去皮关闭蛹和N. 金小蜂鸡蛋将发展正常, 只要他们在一个湿润的会议厅 (培养皿湿滤纸和棉花球), 以防止干燥。在室温下孵化, 从被挫伤的棉球中湿度是足够的, 以防湿度无法控制。</li>
	<li>在 G0胚胎孵化后, 将寄主蛹转移到70% 湿度和 25°C 的新的培养皿中。<ol>
		<li>每日监测寄主蛹并注入 G0幼虫。如果寄主蛹有臭味, 将注入的幼虫转移到新的健康寄主蛹。</li>
	</ol>
	</li>
</ol>
6. 基因组修饰的筛选
<ol>
	<li>使用精美的画笔或胚镐从主机上移除每个金小蜂蛹。注入 G0幼虫应在注射后蛹8天左右。</li>
	<li>将每个被移除的蛹放入一个与棉花连接的隔离玻璃瓶中, 让它们成为 G0成人, 以确保孵出的雌性将会成为有助于下游遗传杂交的处女。 注: 雌性可以从男性的翅膀长度: 女性有较长的翅膀, 延伸过去的身体轮廓时, 转向一侧。所有的微量雌性蛹可以放在一起插入一个小瓶, 同样适用于雄性蛹。</li>
	<li>屏幕 G0个体, 在羽化之后, 通过 PCR 或视觉 (如果扰乱了表型基因)。例如, 如果 CRISPR 用于在给定的基因中创建删除, 而被破坏的基因则赋予一个可见的表型, 然后将个体与受影响的表型 (图 2- viii)21的变种版本进行排序和保持。然而, 如果受影响的基因编码的不表型, 如一个基本基因突变体, 执行交叉方案 (交叉 g0男性与野生型女性, 或交叉 g0女性与野生型男性) 恢复和屏幕可遗传的突变体通过测定致死或不育。 注: 与四腹果蝇类似, N. 金小蜂成人可通过暴露于 CO2来固定, 允许直接操作。此外, 交配雌性会产生100% 单倍体雄性巢, 因此突变的交配雌性可以产生大量的突变雄性, 可用于随后的分析。 注意: 必需基因突变将需要通过交配存活的 G0注入雌性 (想必是杂突变) 保存到野生型雄性;在这个特殊情况下, 一半的 g1的雄性后代应死亡, 由于遗传的致命突变, 而一半的 g1雌性后代将是致命的载体。</li>
	<li>Outcross g0成人和屏幕 g1后代的转基因插入使用转基因标记, 如果目标是转基因。当前转基因仍有待在N. 金小蜂中演示。</li>
</ol>

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H., Loehlin, D. W., Giebel, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+RNAi+in+Nasonia+(parasitoid+wasp).">Larval RNAi in Nasonia (parasitoid wasp).</a> Cold Spring Harb Protoc. (10), (2009).</li><li>Lynch, J. A., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+parental+RNA+interference+in+the+wasp+Nasonia+vitripennis.">A method for parental RNA interference in the wasp Nasonia vitripennis.</a> Nat Protoc. 1 (1), 486-494 (2006).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+heritable+germline+mutations+in+the+jewel+wasp+Nasonia+vitripennis+using+CRISPR/Cas9.">Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9.</a> Sci Rep. 7 (1), 901 (2017).</li><li>Meer, M. M. M., Witteveldt, J., Stouthamer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+microinjection+protocol+for+the+parasitoid+Nasonia+vitripennis.">Development of a microinjection protocol for the parasitoid Nasonia vitripennis.</a> Entomol Exp Appl. 93 (3), 323-327 (1999).</li><li>Werren, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+evolutionary+insights+from+the+genomes+of+three+parasitoid+Nasonia+species.">Functional and evolutionary insights from the genomes of three parasitoid Nasonia species.</a> Science. 327 (5963), 343-348 (2010).</li><li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), 1258096 (2014).</li></ol>

作者没有什么可透露的。

最近的N. 金小蜂基因组的测序已经释放了一个重要的分子工具来描述这个物种中的未知基因的功能,23。CRISPR-Cas9 系统, 和许多其他基因编辑工具, 已证明是有价值的调查基因功能的一些有机体24。然而, 要产生遗传性突变, 这些工具需要执行胚胎 microinjections。因此, 这里演示了一个详细的可视化技术, 其中包括一些允许高效金小蜂胚胎 microinjections 的创新。
总的来说, 这一详细的技术提供了一些重要的创新, 关于现有的方法23, 允许有效的N. 金小蜂胚胎 microinjections。例如, 为了便于快速收集胚胎, 创建了一个产卵工具 (泡沫塞子), 用来限制卵子完全放置在宿主的后端 (图 1), 这极大地促进了大量胚胎的收集很短的一段时间。饲养大量数量的黄蜂的技术也得到了改进和定义。此外, 为了加速胚胎对齐, 胚胎定位装置, 使胚胎能够有效地对齐和注射, 而不必使用双面粘胶带确保胚胎到位 (图 3)。
此外, 通过在注射过程中保持胚胎湿润, 而不干燥胚胎或用油覆盖它们以提高存活率。此外, 还测试了几种毛细管玻璃类型, 并确定了用于构造金小蜂微注射 (表 1,图 4) 的完美针头的参数。此外, 在注射后, 在高 (70%) 室的 pre-stung 宿主中, 胚胎存活率能够显著增加。这些创新使N. 金小蜂的微注射过程更加简化和成功。
根据用户的喜好, 在注射装置和饲养程序方面, 可以对此协议进行细微的修改。但是, 在N. 金小蜂中成功生成突变体的关键步骤将非常重要。例如, 迅速工作, 使注入的胚胎 #62; 1 岁, 确保注射针头足够锋利, 以减少对胚胎的损害, 这两者都是必要的。虽然这个协议应该是有效的产生突变在许多 (如果不是所有) 的基因, 一个主要的限制是 CRIPSR/Cas9 要求的目标 PAM (NGG) 序列, 将决定目标序列。
总之, 这种改进的技术可以用来产生许多种类的基因组修改, 如突变, 删除, 甚至可能使用 CRIPSR/Cas9 技术21, 甚至转基因插入, 以产生转基因N. 金小蜂, 这将大大加速该生物体的功能研究。

宝石黄蜂, N. 金小蜂,是 ectoparasitoids 属的四种物种之一, 它是食肉果蝇, 如 棕 bullata1。由于他们的快速世代周期, 易于饲养在实验室和一系列独特的和重要的生物属性, N. 金小蜂一直是一个重点开发的多种实验工具, 发现在传统的模型生物体.例如, 一些独特的生物属性包括 haplo-二倍体复制系统2, 与微生物和遗传寄生虫的关系3,4, 和一个 supernumary (B) 染色体5,6 ,7。两者结合在一起, 使N. 金小蜂成为一个重要的实验系统, 旨在阐明这些过程的分子和细胞方面, 除了其他包括毒液生产的8,9, 性别确定10,11, 以及轴模式形成的演变和发展12,13,14。此外, 遗传工具箱研究的生物学的N。在过去十年左右的时间里,金小蜂急剧增加, 有了高分辨率基因组的顺序15、几个基因表达研究16、17、18和功能性干扰基因表达的能力依赖于 RNA 干扰 (rna)19,20, 它们一起提高了在这个生物体中执行反向遗传学的可和能力。
尽管在这个有机体中有许多重要的科学进步和扩展的工具箱, 但目前的知识只有一组成功地进行了胚胎 microinjections, 以产生可遗传的基因组修改21。这主要是由于与N. 金小蜂的胚胎的工作困难, 因为它们是相当脆弱和小, 是〜2/3 大小的果蝇胚胎, 使他们一般难以操作。此外, N. 金小蜂雌性将卵放入 pre-stung 苍蝇蛹中, 其中胚胎、幼虫和蛹发育的全部发生。因此, 对于成功的 microinjections, pre-blastoderm 阶段, 必须有效地从寄主蛹收集胚胎, 迅速微量, 并立即移植回其宿主的发展。这些步骤需要精确和灵巧, 以避免损害微量胚胎, 或蛹宿主, 使该技术格外具有挑战性。尽管如此, 十年前发布的一个简短的协议描述了N. 金小蜂胚胎微注射22。然而 , 这个过程要求新的被放置的胚胎燥 , 它使用黏胶带固定蛋为微注射 , 并且不包括技术的视觉示范。因此, 这里描述的是一个更新和修订的协议, 包括一个可视化过程, 详细介绍了一个改进的N. 金小蜂胚胎 microinjections 的 step-by 步协议, 它可以跟随任何基础实验室生成可遗传基因组修改在这个重要模型昆虫。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bugdorm</td>
			<td>Bugdorm</td>
			<td>41515</td>
			<td>Insect Rearing Cage</td>
		</tr>
		<tr>
			<td>Glass Test Tubes</td>
			<td>Fisher Scientific</td>
			<td>982010</td>
			<td></td>
		</tr>
		<tr>
			<td>Flesh fly pupae, Sarcophaga bullata</td>
			<td>Carolina Insects</td>
			<td>144440</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Beveler</td>
			<td>Sutter Instruments</td>
			<td>BV10</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond abrasive plate (0.7u to 2.0u tip sizes)</td>
			<td>Sutter Instruments</td>
			<td>104E</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>Kite R</td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Express programmable microinjector</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000 or P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound Microscope</td>
			<td>Leica DM-750</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminosilicate glass capillary tubing 1mm(outside diameter) X 0.58mm (inner diameter)</td>
			<td>Sutter Instruments</td>
			<td>BF100-58-10</td>
			<td>Can also use Borosilicate or Quartz</td>
		</tr>
		<tr>
			<td>Identi-Plugs</td>
			<td>JAECE</td>
			<td>L800-B</td>
			<td>Foam plugs</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover glass</td>
			<td>VWR</td>
			<td>48366045</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive</td>
			<td>Aron Alpha</td>
			<td>AA471</td>
			<td>For glueing coverslip onto microscope slide</td>
		</tr>
		<tr>
			<td>Fine-tip paintbrush</td>
			<td>ZEM</td>
			<td>2595</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-fine tip forcep</td>
			<td>Fisher Scientific</td>
			<td>16-100-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Femtotips Microloader tips</td>
			<td>Fisher Scientific</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo microinjection and transplantation technique for nasonia vitripennis genome manipulation

宝石黄蜂金小蜂金小蜂已经成为一个有效的模型系统, 用于研究包括性别决定、haplo-二倍体性别测定、毒液合成和宿主-共生相互作用在内的过程。与这个有机体一起工作的一个主要限制是缺乏有效的协议来执行定向基因组的修改。基因组修饰的一个重要部分是通过显微注射将包括 CRISPR/Cas9 分子在内的编辑试剂交付到胚胎中。虽然微注射在许多模型生物体中很好地建立, 但是这种技术在金小蜂中的表现尤其具有挑战性, 主要原因是它的胚胎体积小, 而胚胎发育完全发生在寄生苍蝇蛹。以下过程克服了这些重大挑战, 同时演示了一个简化的, 可视化的程序, 有效地从寄生寄主蛹中去除黄蜂胚胎, microinjecting 它们, 并小心地将它们移植回宿主继续和完成发展。该协议将强有力地增强研究小组在这个生物体内进行高级基因组修饰的能力。

这项协议提供了详细的指导方针的殖民地饲养, pre-blastoderm 胚胎收集, 对齐, 注射, 并随后移植后, 可用于有效的基因组工程在N. 金小蜂。如图 2所示, 成功注射到N. 金小蜂中的一般步骤序列包括: (i) 允许男性和女性成人交配 (〜4天), (二) 提供新鲜的寄主蝇蛹 (S. bullata) 放置在改良的泡沫插头交配雌性和允许产卵 (约45分钟), (iii) 仔细剥离寄生寄主蛹角质层揭露和收集 pre-blastoderm 阶段黄蜂胚胎 (约15分钟), (iv) 对齐收集的胚胎 (约15分钟), (v)microinjecting 基因组修饰成分进入胚胎 (约15分钟), (vi) 小心地将被注射的胚胎放回 pre-stung 宿主以允许适当的发育 (约15分钟), 以及 (vii) 通过转移来防止注射胚胎/宿主的脱水他们进入一个湿润的房间, 大约70% 相对湿度 (约15分钟)。寄生主机, 然后孵化约14天, 以便完全开发的N. 金小蜂胚胎。一旦注射, 成人从主机 (viii), 孤立, 交配, 并筛选他们单独为存在的预期突变。
为了有效的针穿透和显微注射到金小蜂胚胎, 测试了几种类型的毛细管玻璃针, 包括石英、硅酸铝和硼硅酸盐类型。研究发现, 针的质量对于避免注射过程中的破损和堵塞, 以及提高胚胎存活率和转化率都是至关重要的。对于每一个玻璃类型, 一个有效的协议被开发, 以拉针, 以便有一个理想的皮下类似的长尖端有效的N. 金小蜂胚胎注射使用不同的微车夫 (P-1000, 和 P-2000) (表 1, 图 4)。
为了优化这一过程, 在注射不同数量的基因组修饰成分后, 测定存活率。在这里使用的基因组修饰的成分是混合引导 rna 和 Cas9 蛋白 CRISPR 介导的基因组编辑, 这是先前证明在N. 金小蜂21中很好的工作。与先前报告的相似, 这里设计并合成了一个针对朱砂基因的 sgRNA。通过靶向和扰乱这个基因, 一个容易辨认的表型变化在有机体的眼睛颜色被看见19,21。结合各种浓度的 sgRNA (0, 20, 40, 80, 160, 320 ng/µL) 与 Cas9 蛋白 (0, 20, 40, 80, 160, 和 320 ng/µL) 被创建并注入野生类型的胚胎N. 金小蜂。被注射的胚胎的生存率被发现是剂量依赖 (表 2)21。sgRNA 和 Cas9 蛋白浓度的增加导致存活率降低 (表 2), 可能是由于添加了不相干效应。高湿度 (约 70%) 也被认为是重要的胚胎生存后移植到主机, 因为低湿度 (〜 10%) 导致100% 死亡的所有注射胚胎。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56990/56990fig1.jpg"> 
图 1.准备寄主蛹(bullata) 用于金小蜂胚胎产卵.(A)年轻和年老的bullata蛹。较老的蛹有较深的角质层, 而年轻的蛹有更红的色调, 他们的角质层。更年轻的蛹更喜欢产卵的最大化。蛹的后部和前端也可以通过 "后端的火山口状开口" 来区分, 而前端则是一个圆点。(B) 主机 (bullata) 蛹准备为金小蜂胚胎产卵.将寄主蛹插入一个有蛹大小孔的泡沫塞。将寄主蛹的后侧朝内插入, 而0.2 厘米的前端暴露, 允许最大浓度的产卵到前区。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56990/56990fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56990/56990fig2.jpg"> 
图 2.通过显微注射创建金小蜂突变体的时间轴。N. 金小蜂胚集合、CRISPR/Cas9 显微注射和后程序的时间轴。N. 金小蜂允许成人在没有产卵点的情况下交配4天 (i)。下面, 一个新鲜的, 肉蝇寄主蛹, S. bullata, 放置在泡沫塞子内只露出0.5 厘米的后端, 被介绍给妊娠雌性45分钟, 允许寄生 (ii)。同时还制备了注射材料, 包括微注射针头和 CRISPR/Cas9 成分 (iii)。从宿主 (iv) 中收集胚胎, 对齐 (v), 并注入 CRISPR/Cas9 组件 (vi)。注入的胚胎被小心地转移回 pre-stung 寄主 (vii), 并孵育至完全发育 (14 天) (viii)。当成虫出现时, 突变体被筛选为靶基因 (ix) 中预期的 CRISPR/Cas9 诱导突变的表型。整个过程通常需要19天才能完成。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56990/56990fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56990/56990fig3.jpg"> 
图 3.用于衬里胚胎的改良显微镜幻灯片.(a) 片。(B) 显微镜幻灯片。(C) 胚胎对齐装置。片可以粘在显微镜幻灯片上, 用于为注射的胚胎进行排线。片的目的是作为一个边缘, 以便于操作和胚胎的衬里。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56990/56990fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56990/56990fig4.jpg"> 
图 4.注射针准备.(A) 硅酸铝玻璃针的好的和坏的例子。(B) 正直的提示, 正确的斜面, 和斜面的针。采用微拉拔器, 提取硅酸铝玻璃毛细管。生产的针尖, 然后轻轻地打开和提炼使用 beveler。好的斜面针有一个非常尖锐的尖端, 坏斜面针有一个钝尖。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56990/56990fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>毛细管玻璃型</td>
			<td>萨特针牵引器模型</td>
			<td>热</td>
			<td>丝</td>
			<td>速度</td>
			<td>延迟</td>
			<td>拉</td>
			<td>压力</td>
		</tr>
		<tr>
			<td>石英</td>
			<td>P-2000</td>
			<td>750</td>
			<td>4</td>
			<td>40</td>
			<td>150</td>
			<td>165</td>
			<td>-</td>
		</tr>
		<tr>
			<td>铝</td>
			<td>P-1000</td>
			<td>605</td>
			<td>-</td>
			<td>130</td>
			<td>80</td>
			<td>70</td>
			<td>500</td>
		</tr>
		<tr>
			<td>硼</td>
			<td>P-1000</td>
			<td>450</td>
			<td>-</td>
			<td>130</td>
			<td>80</td>
			<td>70</td>
			<td>500</td>
		</tr>
	</tbody>
</table>
表1。针拔器的设置。 注: 拔针器设置因机器而异, 因此每个实验室都需要优化自己的拔针设置。此表已从 Li et al.中修改21
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="3">sgRNA-1</td>
			<td rowspan="3">Cas9</td>
			<td rowspan="3">总胚胎</td>
			<td>移植 (10% 湿度)</td>
			<td colspan="4">移植 (70% 湿度)</td>
		</tr>
		<tr>
			<td>幼虫幸存者</td>
			<td>幼虫幸存者</td>
			<td colspan="3">成年幸存者</td>
		</tr>
		<tr>
			<td>共计 (%)</td>
			<td>共计 (%)</td>
			<td>♂</td>
			<td>♀</td>
			<td>共计 (%)</td>
		</tr>
		<tr>
			<td>无注射</td>
			<td>无注射</td>
			<td>100</td>
			<td>94 (94)</td>
			<td>96 (96)</td>
			<td>66</td>
			<td>26</td>
			<td>92 (92)</td>
		</tr>
		<tr>
			<td>水</td>
			<td>水</td>
			<td>100</td>
			<td>0 (0)</td>
			<td>78 (78)</td>
			<td>44</td>
			<td>32</td>
			<td>76 (76)</td>
		</tr>
		<tr>
			<td>20 ng/µL</td>
			<td>20 ng/µL</td>
			<td>100</td>
			<td>0 (0)</td>
			<td>74 (74)</td>
			<td>34</td>
			<td>34</td>
			<td>68 (68)</td>
		</tr>
		<tr>
			<td>40 ng/µL</td>
			<td>40 ng/µL</td>
			<td>100</td>
			<td>0 (0)</td>
			<td>67 (67)</td>
			<td>30</td>
			<td>32</td>
			<td>62 (62)</td>
		</tr>
		<tr>
			<td>80 ng/µL</td>
			<td>80 ng/µL</td>
			<td>100</td>
			<td>0 (0)</td>
			<td>53 (53)</td>
			<td>24</td>
			<td>22</td>
			<td>46 (46)</td>
		</tr>
		<tr>
			<td>160 ng/µL</td>
			<td>160 ng/µL</td>
			<td>100</td>
			<td>0 (0)</td>
			<td>41 (41)</td>
			<td>16</td>
			<td>22</td>
			<td>38 (38)</td>
		</tr>
		<tr>
			<td>320 ng/µL</td>
			<td>320 ng/µL</td>
			<td>100</td>
			<td>0 (0)</td>
			<td>25 (25)</td>
			<td>10</td>
			<td>10</td>
			<td>20 (20)</td>
		</tr>
	</tbody>
</table>
表2。注射和移植生存和诱变率的基础上注射不同浓度的 sgRNA 和 Cas9。此表已从 Li 修改过et al.21

Watch this Scientific Journal Video about Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation at JoVE.com

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

显微注射的金小蜂金小蜂胚胎是产生遗传基因组改变的必要方法。这里描述的是一个详细的程序, 显微注射和移植的金小蜂金小蜂胚胎, 这将大大促进未来的基因组操纵在这个有机体。

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 ...

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Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

12.2K 次观看.  University of California, Riverside. 显微注射的金小蜂金小蜂胚胎是产生遗传基因组改变的必要方法。这里描述的是一个详细的程序, 显微注射和移植的金小蜂金小蜂胚胎, 这将大大促进未来的基因组操纵在这个有机体。

视频: Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

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

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

转化、基因组编辑和拟合氮的热带大麻树帕拉斯波尼亚安德森

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

以乌苏图病毒为例，验证全基因组纳米孔测序

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

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, ...

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

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

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 ...