Name: microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing
Uploaded: 2018-04-27T14:30:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

这项工作得到了孟山都玉米 Rootworm 知识研究计划的资助, AG/1005 (对勒勒和 YC) 的拨款, 以及从 NCSU 到勒勒的启动资金。FC 得到了孟山都玉米 Rootworm 知识研究计划 (AG/1005) 和国家科学基金会的赠款支持, MCB-1244772 (向勒勒) 授予数字。作者声明没有相互竞争的利益。FC 和勒伊构思并设计了实验;FC、密码和 SP 进行了实验;FC 和勒勒分析了结果;而 FC, NG 和勒伊写的手稿。我们感谢特里萨 O ' 猜疑, 威廉 Klobasa 和斯蒂芬妮 Gorski 为他们的专家协助筛选西铁。我们还感谢韦德博士 (美国农业部, 北中央农业研究实验室, 布鲁金斯, SD) 的运送鸡蛋, 建立实验室殖民地和提供饲养协议。

1. 西铁成人的殖民地级饲养
<ol><li>从可靠的公司或研究实验室获得足够数量的西铁成人 (500-1000), 并在30厘米 3笼中放置。 注意: 强烈建议使用非滞育菌株。</li>
	<li>根据制造商的协议, 准备西铁人工饮食, 并将1厘米厚的层倒入38盎司容器 (请参阅材料表)。混合后, 储存未使用的饮食在摄氏4摄氏度, 长达2月。</li>
	<li>将培养皿 (100 x 15 毫米) 与成人饮食 (10-15 克) 放入30厘米3笼中。当食物低或干燥时, 添加更多的饮食。</li>
	<li>把一个烧瓶 (300 毫升) 与水, 覆盖的棉花球, 这是用来举行一个棉花卷 (6 "x 3/8"), 作为一个水源。当水低的时候, 要改变它。</li>
	<li>保持西铁在26°c 与60% 湿度和14:10 光循环在一个昆虫饲养孵化器。</li>
</ol>2. 胚胎的收集和排列
<ol><li>在不育 100 x 15 毫米2培养皿中使用1% 琼脂在水中制作一个卵子收集室。保存在4摄氏度后琼脂固化。</li>
	<li>要收集新产卵, 请放置一层过滤纸, 然后在琼脂 (图 1A) 表面棉布4层 (每个切口大小)。 注: 棉布可重复使用, 经漂白和蒸压清洗后, 但不需要这样做的第一个用途。过滤纸不应重复使用, 不需要灭菌。</li>
	<li>将蛋收集板放入西铁笼中尽可能晚 (工作日结束), 并盖上锡纸帐篷。一夜之间离开。 注: 从上午10点至上午12点 (14 小时) 的灯延迟产卵, 从而有利于收集较年轻的卵子进行显微注射, 而不需要实验室人员在深夜将收集室放置在笼子里。</li>
	<li>在8或9点左右取出卵子收集室。</li>
	<li>使用镊子在一个时间和地方在烧杯 (500 毫升) 的水中捡起1层棉布。通过轻轻搅拌 (图 1B 和 1C) 从棉布中清洗鸡蛋。</li>
	<li>用灯泡吸管将卵转移到另一个烧杯 (500 毫升) 的水中。重复 2 3x 来清洗鸡蛋。</li>
	<li>使用灯泡吸管将鸡蛋转移到滤纸上 (图 1D), 同时尽量减少水的结转量。</li>
	<li>将黑色滤纸切成小条, 将玻璃滑块和胶带牢牢地放到幻灯片上, 以确保纸张平整 (图 2A)。 注: 黑色滤纸通过减少反射光的量, 有助于提高显微镜下的可见度。</li>
	<li>将无毒胶水 (参见材料表) 应用于细纹的滤纸 (图 2B)。保持胶线比鸡蛋的直径薄, 以避免得到的鸡蛋涂在胶水。</li>
	<li>用细刷子轻轻地将西铁卵从滤纸中移到胶线上。试着把鸡蛋放在胶水上, 在它干掉之前, 保持至少一个鸡蛋的距离。</li>
	<li>将多行鸡蛋放在一张滤纸幻灯片上 (图 3), 从而最大限度地提高注塑效率。</li>
	<li>所有的鸡蛋都放在滤纸上, 等到所有的胶水干了再注射。 注: 对于生殖转换和 CRISPR/Cas9-mediated 基因组编辑, 中午 (下午12点) 注射所有胚胎, 使最老的胚胎不超过19小时。然而, 对于 rna 干扰, 没有时间限制, 因为在西铁, dsRNA 可以在细胞之间移动。事实上, 对于 rna 干扰, 衰老的胚胎可能会导致更好的生存率。</li>
</ol>3. 制备质粒 dna 和注射针
<ol><li>使用无内毒素的商业套件制备高质量的超螺旋质粒 DNA (请参见材料表)。</li>
	<li>准备注射液, 检查每种质粒的浓度, 然后结合辅助质粒 (250 µL 最终浓度), 供体质粒 (750 µL 最终浓度) 和酚红色缓冲 (20% 最终浓度)。涡流解决方案和离心机3分钟的最大速度, 沉淀颗粒, 可能堵塞针。</li>
	<li>使用燃烧/棕色类型的微拉法提取硼硅酸盐玻璃针 (外径1毫米, id 0.58 毫米, 10 厘米长)。用于存储, 安全拉针在双面粘胶带在培养皿或其他明确的容器。 注: 对于微拉拔系统, 使用的设置为: 热 = 335, 滤 = 4, 威 = 40, DEL = 200, 普勒 = 100。</li>
	<li>使用微装载器尖端 (参见材料表) 回填注射针, 吸管 0.5-1.0 µL 在针内, 靠近锥形端。如果发生任何情况, 从针尖上取出气泡。</li>
	<li>将注射针插入针座。</li>
	<li>用细镊子轻轻折断, 或轻轻地触摸/拖动盖玻片或玻璃滑梯上的尖端, 打开针尖。 注: 目标是要有最小的开口, 仍然允许注射混合出来 (较小的开口一般需要更高的注射压力)。斜面针不需要这个步骤, 因为它们已经打开。</li>
</ol>4. 微注射和注射后护理
<ol><li>使用精细的画笔, 小心地湿了 1-3 鸡蛋的表面无菌水, 插入针, 并注入溶液。在表面干燥前将每个卵注入。在注射过程中, 在鸡蛋中寻找少量红色的颜色, 以表示成功的显微注射。粉碎或移除任何看起来受损、空洞或其他无法注射的鸡蛋。 注: 虽然注射压力因针孔大小而异, 但 10-13 psi 是一个很好的起点。</li>
	<li>在所有鸡蛋被注射后, 从玻璃滑梯上取出过滤纸。</li>
	<li>将滤纸放在1% 琼脂盘子的表面上 (如上所述)。</li>
	<li>用塑料石蜡膜封住盘子, 放入26摄氏度的昆虫饲养孵化器中12天。 注: 石蜡膜将有助于防止霉菌之间的交叉污染的菜肴。然而, 任何其他的治疗, 以防止霉菌和细菌的生长可能会减少注射胚胎的生存。</li>
</ol>5. 胚胎的培养和孵化
<ol><li>开始检查胚胎12天后注射的新孵化的幼虫, 并继续每天在未来2周。</li>
	<li>用细刷子将幼虫转移到有发芽玉米和土壤的饲养箱中。 注: 霉菌可能生长在琼脂菜, 但幼虫仍将孵化在大多数情况下。在模具中仔细寻找幼虫来捕捉它们。有些幼虫可能会移到盘子的盖子上, 可能会卡在冷凝中。</li>
	<li>后幼虫在26°c 以下标准西铁养育方法25。</li>
</ol>

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D., 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=piggyBac-based+insertional+mutagenesis+in+Tribolium+castaneum+using+donor/helper+hybrids.">piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids.</a> Insect Mol Biol. 16 (3), 265-275 (2007).</li><li>Gilles, A. F., Schinko, J. B., Averof, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+CRISPR-mediated+gene+targeting+and+transgene+replacement+in+the+beetle+Tribolium+castaneum.">Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.</a> Development. 142 (16), 2832-2839 (2015).</li><li>Phuc, H. K., 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=Late-acting+dominant+lethal+genetic+systems+and+mosquito+control.">Late-acting dominant lethal genetic systems and mosquito control.</a> BMC Biol. 5, 11 (2007).</li><li>Horn, C., Wimmer, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgene-based,+embryo-specific+lethality+system+for+insect+pest+management.">A transgene-based, embryo-specific lethality system for insect pest management.</a> Nat Biotechnol. 21 (1), 64-70 (2003).</li></ol>

作者没有什么可透露的。

虽然已报告9对西铁与 dsRNAs 用于 rna 干扰的目的进行显微注射, 但这是为 microinjecting precellular 西铁胚胎建立最佳做法的第一项议定书, 这是进行生殖改造和/或CRISPR/Cas9 基因组编辑在这个物种。对西铁胚胎的成功显微注射依赖于多种因素, 即转化效率。下面讨论的一些主要问题影响了使用本协议的结果, 在这个甲虫生殖转换。
获得 precellular 胚胎 如上所述, DNA 不会像 dsRNA 那样穿过细胞膜。这意味着重要的是, DNA, mRNA, 和/或蛋白质注射之前, cellularization 发生。因为注射的时机是至关重要的, 所以重要的是要确定一个成功的显微注射的可能的窗口, 根据多长时间 nondiapausing 西铁应变30完成胚胎发生。这是根据对另一甲虫,杂拟谷盗的合胞期胚盘阶段的时间的详细研究计算的。在杂拟谷中, cellularization 发生 ~ 8 h 后卵放置在 30°c31。由于杂拟谷需要3.5 天的时间才能完成30摄氏度的胚胎发生, cellularization 发生在该过程中的第十条路径。由于西铁胚胎需要近2周的发展, cellularization 可能不会发生, 直到第二天后产卵。因此, 据推测, 成功的显微注射的窗口至少有24小时, 这是有益的, 因为西铁需要隔夜产卵, 提供足够数量的卵用于显微注射。虽然此方法用于成功的转基因27, 但可以想象, 通过注入较年轻的胚胎可改善结果, 只要处理早期胚胎不会造成伤害。
DNA 质量和浓度 DNA 浓度在成功的转化过程中扮演关键角色并不奇怪。长期以来, 人们一直认为, 保持质粒 DNA 的最终浓度低于1毫克/毫升是必要的, 以避免潜在的毒性注射胚胎32。虽然现在还不清楚的是, 过去的毒性问题是由于 DNA 本身, 还是由质粒纯化过程中的污染物的结果, 这一经验法则一直坚持了两年。然而, 我们最近发现, 超过1毫克/毫升限制不仅是可能的, 而且这样做可以达到更高的转化率, 导致推测, 没有卵子毒性可能是由于进展的质粒纯化试剂盒,导致污染物减少。鉴于西铁所需的时间, 任何转变率的上升都是有帮助的, 即使这会导致死亡率略高。
微注射前软化卵表面 与杂拟谷不同, 西铁胚胎的外表面 (绒毛膜) 必须在微注射前软化。这是一个关键的步骤, 即使使用石英针。理想情况下, 水应在注射前立即应用于单个胚胎, 但如果在 chorions reharden 前可以完成微量注射, 则可以在2到3个胚胎的组中应用。如果没有这一步, 针一般不会穿透绒毛膜, 从而导致注射尝试失败, 并可能损害针的尖端。此外, 如果针恰好成功地穿透了干绒毛膜, 注射液通常会由于胚胎内的压力高于周围压力而漏出。然而, 它是容易区分干燥从湿 (软化) 胚胎。具体来说, 当干燥时, 胚胎有一个独特的刚性, 圆形, 而添加的水导致他们失去其球形外观。软化不仅使针容易穿透绒毛膜, 而且还能降低胚胎内的压力, 使注射液不漏出。
湿度和模具 在整个胚胎发生过程中, 注入的胚胎在温暖湿润的环境中保存。这些条件不仅为西铁的发展提供了完美的环境, 也促进了模具的生长。在这两个星期期间, 霉菌频繁地生长在琼脂盘子居住西铁胚胎。不幸的是, 胚胎需要高湿度, 并试图减少它的孵化率更低。有趣的是, 霉菌的存在一般对孵化率影响不大, 大多数胚胎孵化时没有问题。此外, 抗真菌治疗对西铁胚胎有害, 因此减少霉菌生长的最佳方法似乎是避免污染;清洁工具, 容器和显微注射系统使用前, 并试图保持时间花 microinjecting 最低, 因为霉菌孢子在许多实验室环境中盛行。
结论 本议定书旨在使更多的研究人员能够在西铁研究中使用转基因技术, 并有望帮助开发更多基于转基因的技术, 用于这种昆虫。在杂拟谷中进行的复杂的基于转基因的实验可能会适合西铁, 包括突变33 和基于 CRISPR 的基因组编辑34。由于这些技术需要对 precellular 胚胎进行显微注射, 这项协议不仅对基于座子的功能基因组研究和/或 CRISPR/Cas9-mediated 基因组编辑, 而且对基于遗传学的害虫管理战略都有帮助。35,36。

西玉米 rootworm (西铁)、Diabrotica virgifera virgifera是玉米的重要害虫1。有趣的是, 西铁似乎比大多数农业害虫更快地克服了控制措施, 因为它们不仅适应生理,而且行为上 2, 3, 4, 5, 6. 在过去的十年中, RNA 干扰 (核糖核酸), 一个强大的功能基因组工具, 已被调查为西铁的潜在控制方法7,8, 并已被用作研究基因功能9的手段。然而, 虽然 rna 干扰是经常执行的双链 RNA (dsRNA) 在其他物种, 注射基 rna 干扰是罕见的西铁。事实上, 只有少数报告通过对 dsRNA 的通过显微注射进入西铁9。原因是西铁可以获得高水平的基因击倒通过摄取 dsRNA7,10, 甚至允许通过喂养 dsRNA 母亲11胚胎效应的研究。这一方法使西铁成为功能基因组分析的一个很好的研究对象,通过rna 干扰, 它减慢了在这个物种的胚胎显微注射方法的发展进展。
尽管有 rna 干扰的能力, 但还是有一些缺点。例如, 并非所有基因对 rna 干扰都一视同仁。这种变异可以使解释功能基因组学的结果更加困难。而且, rna 干扰是瞬态的, 不会产生可遗传的突变。另一方面, 生殖转换, 另一个功能基因组工具, 可以产生可遗传的突变通过插入一个标记的转座因子元素到基因组12, 13.这使得生殖转化成为一种很好的工具, 用于创造突变菌株, 用于长期的基因研究。通过将基因的功能拷贝传递给突变基因组14, 转换也可以用来拯救突变。此外, 生殖转化是多种分子遗传技术的基石。除了敲除和/或拯救基因功能, 生殖转换可用于增强器陷印15, 基因陷印16, Gal4-based 异位表达17, 和基因组范围的突变18。
最近, 已经开发出能够实现复杂基因组编辑的工具。这些工具包括转录激活器类似的效应核酸 (塔伦) 和 CRISPR/Cas9-nuclease 系统19,20。这些新工具的优势在于, 它们为研究人员提供了在几乎任何基因组中几乎任何位置诱导双链 DNA 断裂的能力。一旦诱发, 这些断裂可以通过非同源端连接修复, 这可以引入删除或插入的目标基因, 或通过同源定向修复, 在工程建设的存在, 可以催化替换整个基因或基因区域21,22。然而, 这些方法要求将 DNA、RNA 和/或蛋白质显微注射到非常年轻的胚胎中。
重要的是, 与用于 rna 干扰的 dsRNAs 不同, 用于生殖转化和基因组编辑的核酸和蛋白质不能轻易地穿过细胞膜。因此, 必须在合胞期胚盘阶段(即在昆虫胚 cellularizes 之前) 对质粒 dna、基因和/或蛋白质进行显微注射。这使得注射时机成为关键因素。例如, 在果蝇中, 胚胎需要在卵放置12后两个小时内注射。因此, 研制成功的西铁胚胎微注射方案必须考虑到女性产卵的最佳条件, 以及收集足够数量的 precellular 胚胎的最佳方法。
为了在西铁生物学上引入多种分子遗传工具, 需要开发 precellular 西铁胚胎的收集和 microinjecting 方法。在这里, 我们提供了详细的指示, 连同提示和技巧, 以帮助其他人使用基于转换技术的西铁研究。除了将转基因技术推广到西铁用于功能基因组研究之外, 这些技术还能使强大的新的虫害控制策略, 如基因驱动23,24在这个经济上重要的害虫。

<table><tbody><tr><td>Qualitative Filter Paper (Black)</td><td>Ahlstrom</td><td>8613-0900</td><td>For egg microinjection</td></tr><tr><td>1 oz Containers&amp;nbsp;</td><td>Anny&#039;s Plastic Tableware</td><td>ASET101</td><td>Egg container</td></tr><tr><td>Drosophila Agar Type II</td><td>Apex</td><td>66-103</td><td>Substrate for WCR egg-laying dish</td></tr><tr><td>Featherweight Forceps&amp;nbsp;</td><td>Bioquip</td><td>4748</td><td>Handling larvae and pupae&amp;nbsp;</td></tr><tr><td>Clorox Regular-Bleach1</td><td>Clorox</td><td>44600-30770</td><td>Wash corn and dishes</td></tr><tr><td>Trucker&#039;s Favorite Yellow</td><td>Coor Farm Supply</td><td>502</td><td>Corn for feeding WCR</td></tr><tr><td>Microinjection System (Homemade)</td><td>NA</td><td>Parts &amp;amp; instructions available upon request</td><td>Controls injection pressure (0-20 psi)</td></tr><tr><td>Elmer&#039;s Non-toxic Glue</td><td>Elmer&#039;s Products, Inc.</td><td>E304</td><td>Glue eggs on filter paper</td></tr><tr><td>epTIPS Microloader Tips</td><td>Eppendorf</td><td>C2554691</td><td>Backfilling needle loading tips</td></tr><tr><td>Falcon Tissue Culture Dishes</td><td>Falcon</td><td>25383-103</td><td>Sprouting corn /150 x 25 mm</td></tr><tr><td>Fisherbrand&amp;nbsp;Quantitative-Grade Filter Paper Circles</td><td>Fisher Scientific</td><td>S47576C</td><td>Making agar dish for egg-lay/9cm</td></tr><tr><td>Sparkleen</td><td>Fisher Scientific</td><td>04-320-4</td><td>Wash dishes</td></tr><tr><td>Plain Microscope Slides</td><td>Fisher Scientific</td><td>12-549-3</td><td>Holding filter paper and eggs for microinjection</td></tr><tr><td>Western Corn Rootworm w/o Pollen Substitute</td><td>Frontier Agricultural Sciences&amp;nbsp;</td><td>F9766B</td><td>WCR adult artificial diet</td></tr><tr><td>Cotton Balls, Large</td><td>Genesee Scientific</td><td>51-101</td><td>Close the flask</td></tr><tr><td>Globe Scientific 3.0 mL Small Bulb Transfer Pipettes</td><td>Globe Scientific</td><td>137035</td><td>Collect and transfer eggs</td></tr><tr><td>Leica M165 FC Fluorescence Stereomicroscope&amp;nbsp;</td><td>Leica</td><td>M165 FC&amp;nbsp;</td><td>WCR screening</td></tr><tr><td>DsRed Filter Set for Fluorescence Stereomicroscope</td><td>Leica</td><td>DSR</td><td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td></tr><tr><td>EGFP filter</td><td>Leica</td><td>GFP2</td><td>Excitation filter: 440&amp;ndash;520 nm, emission filter: 510 nm</td></tr><tr><td>BugDorm</td><td>MegaView Science</td><td>DP1000_5P</td><td>Cage for adult colony</td></tr><tr><td>Miniature Paint Brush</td><td>MyArtscape</td><td>MAS-102-MINI</td><td>Liner 2/0&amp;nbsp;</td></tr><tr><td>Joystick Micromanipulator</td><td>Narishige</td><td>MN-151</td><td>Micromanipulator and needle holder</td></tr><tr><td>Microscope XY Stage</td><td>Olympus</td><td>265515</td><td>Microscope stage (adapted for use with stereoscope)</td></tr><tr><td>Grade 90 Cheesecloth</td><td>Online Fabric Store</td><td>CHEE90</td><td>For egg-lay</td></tr><tr><td>Plastic paraffin film&amp;nbsp;</td><td>Pechiney Plastic Packaging</td><td>PM-996</td><td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td></tr><tr><td>Percival Incubator</td><td>Percival&amp;nbsp;</td><td>I41VLH3C8</td><td>WCR growing chamber (insect rearing incubator)</td></tr><tr><td>Narrow Mouth Erlenmeyer Flasks</td><td>VWR</td><td>4980-300-PK</td><td>Water container for adult colony&amp;nbsp;</td></tr><tr><td>Griffin Low Form Beakers</td><td>VWR</td><td>1000-600-PK</td><td>For egg wash</td></tr><tr><td>Braided Cotton Rolls</td><td>Richmond Dental Cotton Co.</td><td>605-3599</td><td>Use in water supply for adult colony&amp;nbsp;</td></tr><tr><td>Petri Dishes (35x10mm)</td><td>SIGMA</td><td>CLS430588-500EA</td><td>For diet plates</td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>143-78-8</td><td>Microinjection buffer</td></tr><tr><td>Laser-based Micropipiette Puller</td><td>Sutter Instrument</td><td>P-2000/G</td><td>Needle puller/Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100</td></tr><tr><td>Premium Topsoil</td><td>The Scotts Company</td><td>71130758</td><td>Soil for cron growing</td></tr><tr><td>Reloc Zippit 2 Mil Zipper Bags</td><td>United States Plastic Corporation</td><td>48342</td><td>Bag agar dish after microinjection/5x7</td></tr><tr><td>Petri Dishes (100x15m)</td><td>VWR</td><td>89038-968</td><td>Making agar dish for egg-lay/ 100 x 15 mm</td></tr><tr><td>6 oz Containers</td><td>Webstaurantstore</td><td>128E506</td><td>WCR single pair mating chamber&amp;nbsp;</td></tr><tr><td>38 oz Containers</td><td>Webstaurantstore</td><td>128NC888</td><td>Larvae rearing box/38 oz</td></tr><tr><td>ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td><td>Webstaurantstore</td><td>128HRD16</td><td>Larvae rearing box/16 oz</td></tr><tr><td>Microinjection Scope&amp;nbsp;</td><td>Wild Heerbrugg</td><td>WILD-M8</td><td>Microinjection scope outfited with an XY stage</td></tr><tr><td>Standard Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td>Microinjection needles</td></tr><tr><td>Adult Western Corn Rootworms</td><td>North Centeral Aqricultural reasearch Laboratory</td><td>Non-diapase strain</td><td>Request from Dr. Bryan Wade French&#039;s lab</td></tr><tr><td>Plasmid DNA Midi Kit</td><td>Qiagen</td><td>12143</td><td>Purification of injection-ready plasmid DNAs</td></tr></tbody></table>

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

西玉米 rootworm (西铁) 是玉米的重要害虫, 以其快速适应害虫管理策略的能力著称。虽然 rna 干扰已被证明是研究西铁生物学的有力工具, 但它有其局限性。具体地说, rna 干扰本身是瞬态的 (即不会导致相关表型的长期孟德尔遗传), 它需要知道目标基因的 DNA 序列。如果兴趣的表型被保守的、甚至是新的基因控制, 那么后者可以被限制, 因为确定有用的目标将是挑战, 如果不是不可能的。因此, 西铁基因组工具箱中的工具数量应该通过开发可用于创建稳定突变株的方法和对西铁基因组进行序列无关的调查来扩大。本文详细介绍了用核酸收集和 microinject precellular 西铁胚胎的方法。虽然此处描述的协议旨在创建转基因西铁, 但 CRISPR/Cas9-genome 编辑也可以使用相同的协议进行, 唯一的区别是注入到胚胎中的溶液的组成。

在开发该议定书时, 一个重要的考虑因素是在显微注射时胚胎发育阶段。具体地说, 生殖转换需要在胚胎 cellularization 之前对 dna 进行显微注射。这是因为 dna 不能轻易地穿过细胞膜。试图通过核染色来可视化西铁胚胎发育的阶段是不成功的, 因为西铁胚胎的 dechorionation 基本上溶解了所有负责胚胎完整性的保护膜。然而, 这一调查线被另一个困难所取代--及时获得足够数量的胚胎。在田野里, 西铁女性把卵放在土壤里, 因此在实验室里, 雌性只在黑暗中产卵是不足为奇的。这使短的白天汇集 (2-4 h) 困难。为了克服这一问题, 一夜间产卵, 并发现产生大量的胚胎。虽然收集 (图 1) 和铺设 (图 2) 胚胎进行显微注射的过程有些费力, 但如果快速完成此协议, 则可以在注射时对 2-19 h 岁之间的胚胎进行显微注射, 而不24小时以上, 如果注射是在当天晚些时候进行。西铁与其他物种的发展时间比较表明, 西铁胚胎仍应 precellular 24 小时后产卵 (见讨论)。
在西铁的生殖改造的第一次尝试涉及共同注射 (图 3) 的帮助 (transposase 源) 和捐助 (座子) 质粒成 precellular 胚胎。捐赠质粒的转化标记是由组成性主动启动子驱动的绿色荧光蛋白 (EGFP) 基因, 由杂拟谷盗( alpha-蛋白启动子 26) 推动。胚胎生存率较低 (表 1), 但26克0成人恢复。为了确定插入到任何生殖细胞中的供体元素, G1胚胎是否被筛选为 EGFP 表达式。虽然荧光后代确实被识别 (图 4), 但用强光源筛选西铁胚胎则被证明是致命的27。下一轮生殖转化利用了一种新的捐赠质粒, 一种是由来自果蝇的组成性主动启动子驱动的红色荧光蛋白 (DsRed) 基因 (热休克蛋白70 启动子 28).虽然存活率得到了很大改善 (表 1), 但幸存的成年人只产生了一个 DsRed 阳性幼虫 (图 5)。然而, 这些实验在两方面特别有用。首先, 发现微注射导致了开发的延迟, 导致了延长的孵化时间。而不是所有胚胎孵化2周内, 注射胚胎孵化在12和26天后, 微注射, 因此需要一个延长监测期, 以确保所有幼仔被收集。第二, 发现新孵化的幼虫相当活跃。他们爬到盖子上, 他们会被卡住, 有时从琼脂盘逃脱。这些问题克服了通过密封培养皿与石蜡膜和检查盖子和琼脂仔细为幼仔。
这一注射协议的最终版本由 microinjecting 西铁胚胎单独使用缓冲区进行测试, 或者使用帮助器和眼睛助剂 (3xP329) 标记供体 DNA 质粒。作为一个额外的控制, 另一组胚胎处理相同, 但没有杏仁。用两种注射液注射四套胚胎。共注射了485个胚胎, 其中有缓冲液和148个胚, 孵化率为 30.5%,表 1)。1450胚胎注射质粒 dna, 289 孵化 (率 = 26.8%)。有趣的是, 在470胚胎中, 接受相同的卵子处理条件, 但没有微量注射, 只有250孵化 (率 = 53.2%)。uninjected 胚胎的存活率比注射胚胎的存活率高出 20%, 这是由于显微注射引起的不可避免的损伤所预期的。然而, 缓冲注射和 dna 注射之间的差别不大, 这表明西铁可以接受添加的 dna 数量 (见讨论)。这些结果也表明, 在早先的努力下, 存活率有所改善。使用此方法成功建立转基因线已在别处报告27。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57497/57497fig1.jpg"> 图 1: 收集卵子。A)卵子收集室。B)放大--在24小时的卵子放置后, 在卵室的一节中缩放。注意, 箭头指示几个鸡蛋的位置。C)在棉布上清洗鸡蛋。D)西铁卵的最终转移。注意, 支架表示有密集的西铁胚胎的区域 (即最小水)。<a href="/files/ftp_upload/57497/57497fig1large.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/57497/57497fig2.jpg"> 图 2: 准备插入幻灯片。a)黑色滤纸紧密贴在标准显微镜幻灯片上。B)在准备过程中, 查看插入幻灯片的某一部分。注意, 箭头 #1 指示一排鸡蛋的位置, #2 部分胶线, #3 用于分散胶水的销。<a href="/files/ftp_upload/57497/57497fig2large.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/57497/57497fig3.jpg"> 图 3: 注入胚胎.八排鸡蛋的注射滑梯。由于注射针的角度, 通过简单的移动阶段可以注射连续的胚胎行。注意注射针中的红色染料大大有助于可视化注射。<a href="/files/ftp_upload/57497/57497fig3large.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/57497/57497fig4.jpg"> 图 4: 转基因 G1胚。从西铁成虫中注射的胚胎, 与piggyBac基于的帮助器和供体质粒作为早期胚胎。明亮的绿色胚胎是积极的增强绿色荧光蛋白 (EGFP) 表达, 而其他不。EGFP 表达式由杂拟谷盗alpha 蛋白启动子26 驱动。<a href="/files/ftp_upload/57497/57497fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57497/57497fig5.jpg"> 图 5: 转基因 G1幼虫.第三龄幼虫, 其父母是以piggyBac为基础的帮助者和捐赠质粒作为早期胚胎共同注射的。在左边的红色发光的幼虫是正面的为 DsRed 表达, 而幼虫在右边不是。DsRed 表达式是由果蝇热休克 70启动子28驱动的。<a href="/files/ftp_upload/57497/57497fig5large.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>实验</td>
			<td>治疗</td>
			<td>鸡蛋</td>
			<td>孵化</td>
			<td>孵化率</td>
		</tr>
<tr>
<td>Exp. #1</td>
			<td>αtubEGFP-捐助者</td>
			<td>910</td>
			<td>57</td>
			<td>6.3%</td>
		</tr>
<tr>
<td>Exp. #2</td>
			<td>hsp70DsRed-donor</td>
			<td>1580</td>
			<td>211</td>
			<td>13.4%</td>
		</tr>
<tr>
<td rowspan="3">Exp. #3</td>
			<td>3xP3EGFP-捐助者</td>
			<td>1450</td>
			<td>389</td>
			<td>26.8%</td>
		</tr>
<tr>
<td>缓冲区</td>
			<td>485</td>
			<td>148</td>
			<td>30.5%</td>
		</tr>
<tr>
<td>无注</td>
			<td>470</td>
			<td>250</td>
			<td>53.2%</td>
		</tr>
</tbody></table>表 1: 微注射数据.从西铁胚胎三微注射试验中孵化率, 显示注射卵子的总数, 成功孵化的幼虫总数, 孵化率。

Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or 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

在这里, 我们提出了收集和 microinjecting precellular 西部玉米 rootworm 胚胎的协议, 目的是执行功能基因组检测, 如生殖转化和 CRISPR/Cas9-genome 编辑。

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 ...

microinjection-western-corn-rootworm-diabrotica-virgifera-virgifera

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

7.4K 次观看.  North Carolina State University. 在这里, 我们提出了收集和 microinjecting precellular 西部玉米 rootworm 胚胎的协议, 目的是执行功能基因组检测, 如生殖转化和 CRISPR/Cas9-genome 编辑。

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

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

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

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

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

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

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

科研

遗传学

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

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

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

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

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

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

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

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

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

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

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

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

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 ...

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

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

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

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

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

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

果蝇中 phiC31 整合酶介导的胚胎转基因

Embryo Microinjection for Transgenesis in Drosophila

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

作者聚焦:优化胚胎显微注射以促进果蝇的转基因

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

胚胎显微注射用于果蝇转基因

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

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

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

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

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

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

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 ...

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

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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

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

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

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

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

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

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

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

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

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

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

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