Name: hyperactive piggybac transposase-mediated germline transformation in the fall armyworm, spodoptera frugiperda
Uploaded: 2021-09-23T14:00:00
Description: Watch this Scientific Journal Video about Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda at JoVE.com

报告的研究得到了美国国家科学基金会I / UCRC，节肢动物管理技术中心的支持，根据资助号IIP-1821936以及行业合作伙伴，农业和食品研究计划竞争性补助金号2019-67013-29351和美国农业部国家粮食和农业研究所（2019-67013-29351和2353057000）。

1. 体 外 合成hyPBase mRNA
注意：合成hyPBase序列的完整编码序列并将其插入pTD1-Cas9载体（见 材料表）以产生pTD1-hyPBase结构，其中包含一个表达hyPBase的盒，T7启动子：polyhedrin-5' UTR：hyPBase：polyhedrin-3' UTR：polyhedrin-3' UTR：poly（A）。pTD1-hyPBase结构的完整序列在 补充材料中提供。
<ol><li>体 外 合成模板的制备<ol>
<li>使用正向引物5'-atgcggtgtgaaataccac...

<ol>
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Y., 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+is+a+flexible+and+highly+active+transposon+as+compared+to+sleeping+beauty,+Tol2,+and+Mos1+in+mammalian+cells.">piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 103 (41), 15008-15013 (2006).</li><li>Tamura, T., 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=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nature Biotechnology. 18 (1), 81-84 (2000).</li><li>Handler, A. M., Harrell, R. 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低转位率和将转基因成分递送到新鲜胚胎中的困难限制了许多非模型昆虫的种系转化的成功，特别是那些来自目鳞翅目昆虫的昆虫。为了提高种系转化率，开发了一种使用最广泛的piggyBac转座酶（hyPBase）的多活性版本7，10。迄今为止，鳞翅目昆虫种系转化的成功主要见于模型昆虫蚕，Bombyx mori11。然而，在其他鳞翅目昆虫?...

秋粘虫（FAW），Spodoptera frugiperda，原产于美国的热带和亚热带地区。目前，这是全球100多个国家的毁灭性昆虫食草动物1。一汽幼虫以350多种寄主植物为食，包括一些重要的主食作物2。一汽成人强大的迁徙能力使其最近从美洲迅速传播到其他地方1，2。因此，这种昆虫现在正威胁着国际上的粮食安全。应用新技术可以促进一汽的高级研究，并提供管理这种害虫的新策略。
昆虫种系转化已被用于研究基因功能并产生转基因昆虫，用于遗传控制3，4。在用于实现昆虫遗传转化的各种方法中，基于piggyBac元素的方法中是最常用的方法5。然而，由于转位率低，在非模型昆虫中进行转基因仍然具有挑战性。最近，开发了一种多活性版本的piggyBac转座酶（hyPBase）6，7。最近第8期在一汽实现了种系转化，这是第一份在鳞翅目昆虫中使用hyPBase的报告。本报告描述了使用hyPBase mRNA产生转基因FAW的详细信息。这里描述的方法可以应用于实现其他鳞翅目昆虫的转化。

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			<td>Narishige</td>
			<td>IM-300</td>
			<td>INJECTION</td>
		</tr>
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			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>INJECTION</td>
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			<td>16004-22</td>
			<td>INJECTION</td>
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			<td>NightSea Full System</td>
			<td>NightSea LLC</td>
			<td>SFA-RB-DIM</td>
			<td>SCREENING</td>
		</tr>
		<tr>
			<td>Nitrogen Gas</td>
			<td>AWG/Scott-Gross</td>
			<td>NI 225</td>
			<td>INJECTION</td>
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			<td>N/A [Request Species/Quantity]</td>
			<td>REARING</td>
		</tr>
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			<td>Spodoptera frugiperda Eggs</td>
			<td>Benzon Research, Inc</td>
			<td>N/A [Request Species/Quantity]</td>
			<td>REARING</td>
		</tr>
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</table>

hyperactive piggybac transposase-mediated germline transformation in the fall armyworm, spodoptera frugiperda

使用转座元件将遗传货物稳定地插入昆虫基因组是功能基因组研究和开发遗传害虫管理策略的强大工具。昆虫转化中最常用的转座元素是 piggyBac，而基于 piggyBac的种系转化已经在模型昆虫中成功进行。然而，在包括农业害虫在内的非模型昆虫中采用这项技术仍然具有挑战性。本文报道了全球农业害虫秋粘虫 （FAW），Spodoptera frugiperda的种系转化，使用过度活跃的 piggyBac 转座酶（hyPBase）。
在这项工作中，生产了hyPBase mRNA，并将其用于胚胎显微注射中的辅助质粒。这一变化导致了转基因FAW的成功诞生。此外，还描述了筛选转基因动物的方法，基于PCR的转基因插入快速检测以及基于热不对称隔行扫描PCR（TAIL-PCR）的整合位点测定。因此，本文提出了一种产生转基因FAW的方案，这将促进FAW和其他鳞翅目昆虫中基于 piggyBac的转基因。

一种表达含hyPBase的T7启动子的构建体：polyhedrin-5'UTR：hyPBase：polyhedrin-3'UTR：产生聚（A）信号（图1A）并扩增为〜2.2 kb PCR片段以在体外合成hyPBase mRNA（图1B）。然后，产生hyPBase mRNA并进行琼脂糖凝胶电泳。在1%琼脂糖凝胶上检测到预期大小（〜1.1 kb条带）的mRNA（图1C）。
然后，制备基于 piggyBac的EGF...

Watch this Scientific Journal Video about Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda at JoVE.com

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

秋粘虫 Spodoptera frugiperda的种系转化是使用过度活跃的 piggyBac 转座酶的mRNA实现的。

多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化

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

该协议显示了使用基于piggyBac的转化系统生产转基因秋粘虫。我们希望这些方法将有利于改善转化，不仅在秋粘虫中，而且在鳞翅目昆虫中。这里描述的方法使鳞翅目害虫昆虫的种系转化效率很高。演示该程序的将是我实验室的博士后研究员Xien Chen博士。首先将12只雄性蛹和12只雌性蛹放在一个盒子里。将10%蔗糖溶液放入盒子中喂养成人。用纸巾盖住盒子。在闭合后的第二天，将成虫暴露在强光下过夜。在关闭后的第三天，将成年人转移到黑暗的地方。在过度放置后两小时内收集胚胎。在纸巾上加入一滴水，将新鲜鸡蛋放在冰上的培养皿中。用细刷子将鸡蛋轻轻地转移到载玻片上，并在载玻片上逐个对齐。在显微注射之前，保持载玻片，将鸡蛋放在冰上。使用自动显微注射器的补偿压力，将每微升200纳克小猪Bac表达质粒，每微升200纳克超活性猪Bac转座酶mRNA和无菌蒸馏水的混合物注入鸡蛋中。将注射的卵保持在27加或零下1摄氏度，60±10%相对湿度，直到孵化。用人工饲料喂养孵化的幼虫，并在早期凝乳和星形阶段将每个幼虫转移到一个小杯子中。收集蛹，并将大约150只雌性和150只雄性蛹放在笼子里。在笼子里挂几条30厘米×15厘米大小的纸巾，以收集鸡蛋。每天收集带有鸡蛋的纸巾，并将鸡蛋保持在适当的条件下直到孵化。将新生儿幼虫保持在四摄氏度下五分钟以使其固定，然后将其转移到冰冷的盘子上。在荧光体视显微镜下对固定的新生儿幼虫进行筛查。选择EGFP阳性的新生儿，在大约两周内以27摄氏度或零下一摄氏度将其提升到蛹阶段。使用1%琼脂糖凝胶检测体外合成的过度活跃的piggyBac转座酶mRNA。PDS注射的胚胎没有显示EGFP信号，而表达EGFP的质粒和过度活跃的piggyBac转座酶mRNA注射的胚胎显示出EGP荧光，表明piggyBac构建是成功的。从G0成虫自交产生的卵中获得的G1幼虫显示出强烈的EGFP信号。使用从EGFP阳性幼虫中分离的基因组DNA作为模板，成功扩增了含有启动子和EGFP片段的片段。然而，当从野生型幼虫中分离出基因组DNA作为模板时，没有检测到相同的片段。用于显微注射的卵子应尽可能新鲜，并且在显微注射之前必须始终保持在冰上以减缓其发育。该技术为研究人员回答害虫功能基因组学中的问题并为这种全球害虫开发遗传控制方法铺平了道路。

Research

JoVE Journal

Biology

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

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

脑片的刺激，利用微流控网络和标准灌注腔

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

高效的聚乙二醇（PEG）介导的转化苔小立碗藓</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

的代谢活性细菌在通才的肠道鉴定<em&gt;海灰翅夜蛾</em&gt;通过DNA的稳定同位素探测使用<sup&gt; 13</sup&gt; C-葡萄糖

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

睾丸融合的显微外科梗阻

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

用于黑腿蜱、肩突硬蜱基因编辑的胚胎注射技术

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

秀丽隐杆线虫种系内突触复合体的超分辨率显微镜

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...