Name: microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing
Uploaded: 2018-04-27T14:30:00
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?...

<ol>
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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 ?...

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 border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Qualitative Filter Paper (Black)</td>
			<td style="width:189px;">Ahlstrom</td>
			<td style="width:287px;">8613-0900</td>
			<td style="width:339px;">For egg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 oz Containers&#160;</td>
			<td style="width:189px;">Anny&#39;s Plastic Tableware</td>
			<td>ASET101</td>
			<td>Egg container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td style="width:189px;">Apex</td>
			<td style="width:287px;">66-103</td>
			<td>Substrate for WCR egg-laying dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight Forceps&#160;</td>
			<td>Bioquip</td>
			<td style="width:287px;">4748</td>
			<td>Handling larvae and pupae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clorox Regular-Bleach1</td>
			<td style="width:189px;">Clorox</td>
			<td style="width:287px;">44600-30770</td>
			<td>Wash corn and dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trucker&#39;s Favorite Yellow</td>
			<td>Coor Farm Supply</td>
			<td>502</td>
			<td>Corn for feeding WCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microinjection System (Homemade)</td>
			<td style="width:189px;">NA</td>
			<td style="width:287px;">Parts &#38; instructions available upon request</td>
			<td>Controls injection pressure (0-20 psi)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Elmer&#39;s Non-toxic Glue</td>
			<td>Elmer&#39;s Products, Inc.</td>
			<td style="width:287px;">E304</td>
			<td>Glue eggs on filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">epTIPS Microloader Tips</td>
			<td style="width:189px;">Eppendorf</td>
			<td style="width:287px;">C2554691</td>
			<td>Backfilling needle loading tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Tissue Culture Dishes</td>
			<td style="width:189px;">Falcon</td>
			<td>25383-103</td>
			<td>Sprouting corn /150 x 25 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand&#160;Quantitative-Grade Filter Paper Circles</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td>S47576C</td>
			<td>Making agar dish for egg-lay/9cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sparkleen</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">04-320-4</td>
			<td>Wash dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plain Microscope Slides</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">12-549-3</td>
			<td>Holding filter paper and eggs for microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Western Corn Rootworm w/o Pollen Substitute</td>
			<td style="width:189px;">Frontier Agricultural Sciences&#160;</td>
			<td>F9766B</td>
			<td>WCR adult artificial diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton Balls, Large</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td>Close the flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Leica M165 FC Fluorescence Stereomicroscope&#160;</td>
			<td style="width:189px;">Leica</td>
			<td>M165 FC&#160;</td>
			<td>WCR screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DsRed Filter Set for Fluorescence Stereomicroscope</td>
			<td style="width:189px;">Leica</td>
			<td>DSR</td>
			<td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGFP filter</td>
			<td style="width:189px;">Leica</td>
			<td>GFP2</td>
			<td>Excitation filter: 440&#8211;520 nm, emission filter: 510 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BugDorm</td>
			<td>MegaView Science</td>
			<td>DP1000_5P</td>
			<td>Cage for adult colony</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Miniature Paint Brush</td>
			<td style="width:189px;">MyArtscape</td>
			<td>MAS-102-MINI</td>
			<td>Liner 2/0&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Joystick Micromanipulator</td>
			<td style="width:189px;">Narishige</td>
			<td style="width:287px;">MN-151</td>
			<td>Micromanipulator and needle holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Microscope XY Stage</td>
			<td style="width:189px;">Olympus</td>
			<td style="width:287px;">265515</td>
			<td style="width:339px;">Microscope stage (adapted for use with stereoscope)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grade 90 Cheesecloth</td>
			<td style="width:189px;">Online Fabric Store</td>
			<td>CHEE90</td>
			<td>For egg-lay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic paraffin film&#160;</td>
			<td style="width:189px;">Pechiney Plastic Packaging</td>
			<td>PM-996</td>
			<td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Percival Incubator</td>
			<td style="width:189px;">Percival&#160;</td>
			<td style="width:287px;">I41VLH3C8</td>
			<td>WCR growing chamber (insect rearing incubator)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Mouth Erlenmeyer Flasks</td>
			<td style="width:189px;">VWR</td>
			<td>4980-300-PK</td>
			<td>Water container for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Griffin Low Form Beakers</td>
			<td style="width:189px;">VWR</td>
			<td>1000-600-PK</td>
			<td>For egg wash</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Braided Cotton Rolls</td>
			<td>Richmond Dental Cotton Co.</td>
			<td>605-3599</td>
			<td>Use in water supply for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (35x10mm)</td>
			<td>SIGMA</td>
			<td>CLS430588-500EA</td>
			<td>For diet plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol Red</td>
			<td>Sigma</td>
			<td style="width:287px;">143-78-8</td>
			<td>Microinjection buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Premium Topsoil</td>
			<td>The Scotts Company</td>
			<td style="width:287px;">71130758</td>
			<td>Soil for cron growing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Petri Dishes (100x15m)</td>
			<td style="width:189px;">VWR</td>
			<td style="width:287px;">89038-968</td>
			<td>Making agar dish for egg-lay/ 100 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128E506</td>
			<td>WCR single pair mating chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">38 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128NC888</td>
			<td>Larvae rearing box/38 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128HRD16</td>
			<td>Larvae rearing box/16 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microinjection Scope&#160;</td>
			<td style="width:189px;">Wild Heerbrugg</td>
			<td style="width:287px;">WILD-M8</td>
			<td style="width:339px;">Microinjection scope outfited with an XY stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>Microinjection needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Adult Western Corn Rootworms</td>
			<td style="width:189px;">North Centeral Aqricultural reasearch Laboratory</td>
			<td style="width:287px;">Non-diapase strain</td>
			<td>Request from Dr. Bryan Wade French&#39;s lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plasmid DNA Midi Kit</td>
			<td style="width:189px;">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?...

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 ...

Research

JoVE Journal

Bioengineering

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

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

科研

生物工程

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

用于全基因组 DNA 分析的超长读数测序

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

CRISPR/Cas12a 多路复用基因组编辑糖母细胞和酵母像素艺术的创作

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

测量超极化 [1-13C]硫化的旋转-莱迪兹松弛磁场依赖性

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

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

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...