Name: applications of rna interference in american cockroach
Uploaded: 2021-12-17T15:00:00
Description: Watch this Scientific Journal Video about Applications of RNA Interference in American Cockroach at JoVE.com

本研究由国家自然科学基金（32070500、31330072 31620103917、31572325、广东省自然科学基金（批准号：2021B1515020044和2020A1515011267）、广东省科学技术厅（批准号：2019B090905003和2019A0102006）、广州市科学技术厅（批准号：202102020110号）资助， 获深圳市科技计划（助学金编号：KQTD20180411143628272 to Sh.L.）。

P. americana的系列最初由Huiling Hao博士提供。该物种已经进行了30年的近亲繁殖9。
1. 美洲鹦鹉的孵化和投饲
<ol><li>收集美洲 鹦 鹉的新鲜卵母（产卵后立即），并在25°C和60%湿度的黑暗培养箱中孵育约25天。然后在孵化前3天将温度和湿度提高到30°C和75%。</li>
	<li>使用具有4毫米孔径的筛子将孵化的若虫与卵形目分开。</li>
	<li>将...

<ol>
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V., 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=EAG+channels+expressed+in+microvillar+photoreceptors+are+unsuited+to+diurnal+vision.">EAG channels expressed in microvillar photoreceptors are unsuited to diurnal vision.</a> The Journal of Physiology. 595 (16), 5465-5479 (2017).</li><li>Li, S., 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=The+genomic+and+functional+landscapes+of+developmental+plasticity+in+the+American+cockroach.">The genomic and functional landscapes of developmental plasticity in the American cockroach.</a> Nature Communications. 9 (1), 1008 (2018).</li><li>Zhao, Z., 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=Grainy+head+signaling+regulates+epithelium+development+and+ecdysis+in+Blattella+germanica.">Grainy head signaling regulates epithelium development and ecdysis in Blattella germanica.</a> Insect Science. 28 (2), 485-494 (2021).</li><li>Lozano, J., Belles, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+repressive+function+of+Kr&#252;ppel+homolog+1+on+insect+metamorphosis+in+hemimetabolous+and+holometabolous+species.">Conserved repressive function of Kr&#252;ppel homolog 1 on insect metamorphosis in hemimetabolous and holometabolous species.</a> Scientific Reports. 1, 163 (2011).</li><li>Philip, B. N., Tomoyasu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+knockdown+analysis+by+double-stranded+RNA+injection.">Gene knockdown analysis by double-stranded RNA injection.</a> Methods in Molecular Biology (Clifton, N. J). 772, 471-497 (2011).</li><li>Zheng, 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=CRISPR+interference-based+specific+and+efficient+gene+inactivation+in+the+brain.">CRISPR interference-based specific and efficient gene inactivation in the brain.</a> Nature Neuroscience. 21 (3), 447-454 (2018).</li><li>Garbutt, J. S., Bell&#233;s, X., Richards, E. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+double-stranded+RNA+in+insect+hemolymph+as+a+potential+determiner+of+RNA+interference+success:+Evidence+from+Manduca+sexta+and+Blattella+germanica.">Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica.</a> Journal of Insect Physiology. 59 (2), 171-178 (2013).</li><li>Parrish, S., Fleenor, J., Xu, S., Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+a+dsRNA+trigger:+differential+requirement+for+the+two+trigger+strands+in+RNA+interference.">Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference.</a> Molecular Cell. 6 (5), 1077-1087 (2000).</li><li>Lemonds, T. R., Liu, J., Popadi&#263;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+the+melanin+pathway+to+overall+body+pigmentation+during+ontogenesis+of+Periplaneta+americana.">The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana.</a> Insect Science. 23 (4), 513-519 (2016).</li><li>Jackson, A. L., Linsley, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+amidst+the+silence:+Off-target+effects+of+siRNAs.">Noise amidst the silence: Off-target effects of siRNAs.</a> Trends in Genetics: TIG. 20 (11), 521-524 (2004).</li><li>Patel, M., Peter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DISE-inducing+shRNAs+by+monitoring+cellular+responses.">Identification of DISE-inducing shRNAs by monitoring cellular responses.</a> Cell Cycle (Georgetown, Tex). 17 (4), 506-514 (2018).</li><li>Vent&#243;s-Alfonso, A., Ylla, G., Monta&#241;es, J. C., Belles, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNMT1+promotes+genome+methylation+and+early+embryo+development+in+cockroaches.">DNMT1 promotes genome methylation and early embryo development in cockroaches.</a> iScience. 23 (12), 101778 (2020).</li><li>Wang, 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=Variation+in+RNAi+efficacy+among+insect+species+is+attributable+to+dsRNA+degradation+in+vivo.">Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo.</a> Insect Biochemistry and Molecular Biology. 77, 1-9 (2016).</li><li>Bi, F., Liu, N., Small Fan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interfering+RNA:+A+new+tool+for+gene+therapy.">interfering RNA: A new tool for gene therapy.</a> Current Gene Therapy. 3 (5), 411-417 (2003).</li></ol>

该报告描述了 美洲猪笼草的方法学循序渐进的RNAi策略;值得注意的是，它也可以应用于其他蟑螂（例如Blattella germanica ）和许多其他变化很小的昆虫。然而，RNAi的基因沉默效率并不总是足够高，与基因敲除策略13相比，存在明显的劣势。基因水平的以下残留效应可能会干扰真正的表型。为了确保RNAi治疗的成功，需要考虑几个基本条件，包括动物的身体状况，对照组?...

RNA干扰（RNAi）是一种进化保守的机制，自Andrew Fire和Craig Mello2开发出双链RNA（dsRNA）介导的基因沉默策略以来，逐渐成为抑制许多生物体1中基因表达的重要反向遗传工具。dsRNA被细胞中的DsRNA切割成21-23个核苷酸的片段，小干扰RNA（siRNA），由细胞中的Dicer酶激活RNAi途径。然后将siRNA掺入RNA诱导的沉默复合物（RISC）中，该复合物与靶mRNA偶联，导致mRNA裂解，最终导致基因功能丧失3，4，5。在昆虫物种中，到目前为止，许多系统RNAi实验已经在许多昆虫目中报道，例如直翅目，等翅目，半翅目，鞘翅目，神经翅目，双翅目，膜翅目，鳞翅目和Blattodea5，6，7，8。
蟑螂（Blattaria）是发育和变质研究中必不可少的昆虫家族，其生长周期快，对环境适应性强，发育可塑性高9。在发现RNAi与蟑螂兼容之前，由于蟑螂遗传操纵技术的稀缺，以前的研究仅关注蟑螂的预防和控制。蟑螂卵子的独特结构使得使用CRISPR-Cas9系统进行基于胚胎注射的基因敲除具有挑战性。此外，蟑螂中的大多数组织（如美洲猪笼草）表现出强大的全身RNAi反应，允许通过注射一个或多个靶向dsRNA9，10，11来快速产生功能失调的表型。这些特征使RNAi成为美洲猪笼草基因功能研究中不可或缺的技术。
尽管已经报道了RNAi在 美洲猪 笼草功能基因研究中的应用，但没有详细或逐步的描述。本报告为 美洲蟑螂中的RNAi提供了一个分步操作指南，可用于其他蟑螂的基因功能研究。此外，本指南不仅限于Blattodea，还可以应用于许多其他昆虫，只需稍作修改即可。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>701 N 10 &#181;L Syr (26s/51/2)</td>
			<td>Hamilton</td>
			<td>PN:80300</td>
			<td>Injection</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ningbo Jiangnan Instrument Factory</td>
			<td>RXZ-380A-LED</td>
			<td>For cockroaches hatching and feeding</td>
		</tr>
		<tr>
			<td>Micro-injection pump</td>
			<td>Alcott Biotechnology</td>
			<td>ALC-IP600</td>
			<td>Injection</td>
		</tr>
		<tr>
			<td>pTOPO-Blunt Cloning Kit</td>
			<td>Aidlab Biotechnology</td>
			<td>CV16</td>
			<td>For Gene clonging</td>
		</tr>
		<tr>
			<td>quantitative Real-Time PCR Systems</td>
			<td>Bio-Rad</td>
			<td>CFX Connect</td>
			<td>For qRT-PCR analysis</td>
		</tr>
		<tr>
			<td>T7 RiboMAX Express RNAi System</td>
			<td>Promega</td>
			<td>P1700</td>
			<td>For dsRNA synthesis, which contains Rnase A Solution (4 &#956;g/&#956;L), Sodium Acetate, 3.0M (pH 5.2), Enzyme Mix, T7 Express, Nuclease-Free water, Express T7 2x Buffer, RQ1 RNase-Free DNase</td>
		</tr>
		<tr>
			<td>Thermal Cyclers</td>
			<td>Bio-Rad</td>
			<td>S1000</td>
			<td>For DNA amplification</td>
		</tr>
	</tbody>
</table>

applications of rna interference in american cockroach

蟑螂是一种卫生害虫，由于其易于觅食和半代谢特性，是昆虫发育和变质研究中必不可少的物种。加上注释良好的基因组序列，这些优势使得美洲蟑螂， 美洲角行星， 成为重要的半代谢昆虫模型。受敲除策略不足的限制，基于有效RNA干扰（RNAi）的基因敲低成为 美洲桫椤功能基因研究中不可或缺的技术。本方案描述了 美洲猪笼草的RNAi操作技术。该方案包括（1）在适当的发育阶段选择 美洲疟原虫 ，（2）注射设置的准备，（3）dsRNA注射和（4）基因敲低效率检测。RNAi是 美洲猪笼草中一种强大的反向遗传工具。大多数 美洲疟原虫 组织对细胞外dsRNA敏感。它的简单性使研究人员能够在一次或多次靶向dsRNA注射下快速获得功能失调的表型，使研究人员能够更好地利用 美洲疟原虫 进行发育和变质研究。

图1 显示了成功的注射。带有微直径针头的显微注射注射器应水平放置在加强器上（图1A）。针 通过 两个腹部索米特之间的间隙水平插入表皮（图1B）。确保液体进入 美洲猪笼 草腹部。针头太陡峭的角度会损害内脏器官（图1C），注射不当导致dsRNA溶液从腹部泄漏出来（图1D</stro...

Watch this Scientific Journal Video about Applications of RNA Interference in American Cockroach at JoVE.com

Applications of RNA Interference in American Cockroach

本方案描述了 美洲蚴中RNAi操作技术的分步指南。

RNA干扰在美洲蟑螂中的应用

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

该协议提供了一种研究美洲蟑螂的简单方法。一种常见的卫生害虫，也是中药的益虫。它将有助于通过敲除特定基因来探索美洲蟑螂多种生命活动的分子机制。这项技术的主要优点是它可以敲除美洲蟑螂或其他不适合基因编辑的类似昆虫中的基因。该技术可用于探索各个领域，例如遗传和表观遗传调节，组织的发育过程，附属物的再生，交配行为以及美洲蟑螂的其他有趣方面。该技术易于操作且用途广泛。我们建议在DSRA注射治疗期间保持动物健康。我相信新手在几次练习后可以轻松掌握这种技术。首先，用四毫米孔径筛将孵化的若虫与卵形目分开。用玻璃管挑出新鲜出白色的南美白苣苔。将P.americana放入新容器中，等待正确的治疗阶段。在反应体系中，加入10微升T7 2X缓冲液，2微升T7表达酶混合物和2微克PCR产物。最后，用双蒸馏水将总体积补足至20微升。手动轻轻倒置混合，在 37 摄氏度下孵育 30 分钟，然后在 70 摄氏度下孵育 10 分钟。以1至200的比例用DEPC水稀释每微升RNase A溶液4微克。然后向系统中加入1微升每微升1单位RQ1 RNase游离DNASe和1微升稀释的RNase A溶液。在37摄氏度下孵育30分钟。然后加入总体积的10%的乙酸钠和三体积的异丙醇总体积。手动轻轻倒置混合，然后放在冰上五分钟，在4摄氏度下以13， 000克离心10分钟。用移液器除去上清液。接下来，用75%乙醇洗涤残余物，用25%DEPC处理过的水，然后在4摄氏度下以13， 000g离心5分钟。再次除去上清液，在室温下风干沉淀15分钟。然后用约100微升的DEPC水溶解双链RNA，并将其稀释至每微升2微克的最终浓度。在显微注射泵中设置程序，以确保每次注射的体积一致。然后用DEPC水填充注射器8至10次来清洁注射器。将10微升注射器安装在微型注射泵上。然后启动泵，用10微升双链RNA溶液填充注射器。用二氧化碳麻醉蟑螂，然后用镊子轻轻地捡起它们，用手将蟑螂送到针头上。接下来，通过两个腹部索米特之间的间隙水平插入针头，以对抗表皮。将双链RNA溶液注入蟑螂中，确保针尖尽可能靠近表皮，以避免损伤内脏。最后，拔出针尖。将注射的蟑螂放入干净的生物测定容器中。等待大约10到20分钟，让他们从二氧化碳的影响中恢复过来。用注射日期，双链RNA的类型和剂量以及美洲蒿草的年龄标记容器。选取Ddc或DOPA脱羧酶和Dpp或十五淀粉基因作为两个实例，观察双链RNA注射后麦芽蟑螂的表型，以动物无靶标的dsMocK为阴性对照。采用qRT PCR检测RNAi效率。基因被显著敲低，dsDdc的P值小于0.01，dsDpp的P值小于0.05。具有敲低Ddc基因的P.americana由于黑化阻滞而具有白色表皮。在dsDpp注射的实验中，RNAi破坏了肢体再生。昆虫应该是健康的，注射时不会造成伤害。这项技术提供了一种简单的方法来探索无法进行基因编辑的昆虫的基因功能，例如美洲蟑螂。

Research

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Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

用于细胞培养和皮肤应用的薄膜复合硅弹性体: 制造和表征

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...