Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

1.蚊种和饲养<ol><li>维持A.冈比亚 G3和A.伊蚊利物浦IB12菌株（在下面的代表性研究中使用），或者根据标准的实验室实践或如先前23,24描述的其它感兴趣的菌株。 </li></ol> 2.双链RNA和siRNA设计与制作<ol><li>设计引物来构造特定的感兴趣的基因长的dsRNA模板。使用E-的RNAi工具25。根据选择或作为该方法产生的dsRNA先前所述19。 <ol><li>对于阴性对照，合成的dsRNA 19对应于绿色荧光蛋白（GFP），β半乳糖苷酶（β-GAL）或一些不表达于蚊子其他基因的序列。如果需要的话，19的dsRNA用于生成摘要如下代表性的结果可被用作阳性对照。商店的dsRNA溶解在无RNA酶的水在-80℃下直到需要。 </li></ol></li><li>或者，根据标准实验室实践设计基因特异性siRNA或如前面10所描述。加扰击倒的siRNA的序列来设计阴性对照siRNA不对应于任何蚊基因。购买定制的siRNA，这些都可以通过一些信誉良好的供应商。 <ol><li>如果需要的话，siRNA的20,21用于获得下面总结了代表性的结果可被用作阳性对照。商店的siRNA溶解在无RNA酶的水在-80℃直到需要。 </li></ol></li></ol> 3.准备壳聚糖/干扰RNA纳米粒子<ol><li>收集预制备的RT 100mM的硫酸钠（100毫用Na 2 SO 4在去离子H 2 O）和0.1M乙酸钠（0.1M NAC 2 H 3 O 2 -0.1 1M乙酸，pH 4.5的去离子H 2 O）缓冲区。 </li><li>壳聚糖溶解（​​≥75％脱乙酰化）的0.1M乙酸钠缓冲液，使0.02％（重量/体积）的工作溶液，并在使用前保持该溶液在RT。 </li><li>溶解的dsRNA或siRNA在50μl去离子H 2 O和它添加到100mM的硫酸钠缓冲剂使32微克每100微升的50mM硫酸钠的dsRNA / siRNA的的100微升溶液中。 </li><li>加入100微升的脱乙酰壳多糖溶液，以所述dsRNA / siRNA的溶液中，然后加热的水浴中的混合物在55℃下1分钟。设置了控制通过加入100微升的50mM硫酸至100μl的脱乙酰壳多糖溶液的钠，并按照相同的程序。 </li><li>立即混合溶液30秒由高速涡流在RT以促进纳米粒子的形成。 </li><li>在13000×g离心离心混合物10分钟，在RT下，在这之后时间的颗粒应该是可见的。将上清液转移到一个新的1.5毫升管。用它来制备蚊子食前空气干燥沉淀在室温≈10分钟。 </li><li> M通过使用上清液从控制（见3.5）作为空白来计算的dsRNA / siRNA的那保留在上清液中的总量easure的dsRNA / siRNA对上清液中的浓度。使用的dsRNA / siRNA的的起始量和残留在上清液来计算的dsRNA / siRNA对截留在纳米颗粒的百分比的量之间的差。这装载效率通常在90％以上。 </li><li>如果需要更多的纳米粒子重复相同的过程。立即用干的纳米粒子。之前使用的颗粒冷库的影响尚未评估。 </li></ol> 4.准备蚊子食物含壳聚糖/干扰RNA纳米粒子<ol><li>制备1ml的2％的琼脂糖溶液（重量/体积）在去离子水中，熔化琼脂糖，并保持熔化的琼脂糖溶液在55℃的水浴后使用。 </li><li>混合鱼食品薄片（47％的​​粗蛋白，最小值粗脂肪10％，最大粗纤维3％）和干酵母在1（重量/重量）：2的比例。研磨该混合物以用研钵和研杵（及格通过50号美国标准试验筛）的小颗粒。使用地面食品，这应该是带褐色的颜色，或者立即或者在密封容器中储存于4℃几周。 </li><li>在1.5ml管中，混合6毫克地面食品与来自第3.7节用牙签将干燥的纳米颗粒。 </li><li>加入30微升2％的预熔化的琼脂糖凝胶溶液的食品的纳米粒子混合物;用牙签或枪头立即，充分搅拌。 </li><li>使用含有食品和纳米颗粒立即喂养蚊子幼虫的凝胶。可替换地，一旦将凝胶在室温下完全固化，储存于4℃的凝胶，并使用第二天，或在-80℃以备后用。 </li></ol> 5.饲养蚊子幼虫食含壳聚糖/干扰RNA纳米粒子<ol><li>喂养A.冈比亚蚊子幼虫： <ol><li>删除SI从1.5 ml管使用牙签切成使用干净的刀片或牙签6等于切片ngle凝胶颗粒。 </li><li>转让20第三龄幼虫到含有100毫升去离子水的500毫升的培养皿。 </li><li>加入每一天四天培养皿一次凝胶颗粒（切碎成小块）的一个切片喂蚊子幼虫。请务必遵守幼虫取食的颗粒，应在尺寸显著减少或第二天完全不存在。时间之后，蚊子就会发展成晚期第四龄幼虫。 </li><li>记录在实验过程中的任何明显的表型变化。审查的转录水平和其他表型的改变为在第6在四个天期间的结束讨论。 </li></ol></li><li>喂养A.伊蚊孑孓： <ol><li>切凝胶粒料成使用干净的刀片或牙签6等于切片。 </li><li>卵孵化后，科幻放置50岁同步24小时首先龄幼虫成≈40毫升去离子水的培养皿。 </li><li>饲料每培养皿中的幼虫一个片4小时/天，然后回到幼虫转移到正规的幼虫饮食的2：1地面鱼食片和干酵母一天的休息。每天在整个4龄幼虫重复上述步骤。 </li><li>检查转录水平和其他表型的改变，如第6，在预期的发育时间点讨论。 </li></ol></li></ol> 6.确认基因敲除的<ol><li> A.在通过定量RT-PCR相对定量成绩单伊蚊和A.冈比亚幼虫。 <ol><li>执行与如上所述11,19,20，或按照标准实验室程序在至少10汇总控制与实验幼虫分析定量RT-PCR测定具有三个生物学重复。代表性的结果包括在下面。 </li><li>对于特定的组织类型/身体部分（ 即 ，脑或天线），射孔的分析RM QRT-PCR如6.1.1以下解剖描述来恢复感兴趣的组织（例如，参见迈索尔等人 21）。 </li></ol></li><li>通过原位杂交确认击倒<ol><li>合成地高辛标记的反义，并根据标准的实验室实践或描述26感知控制的核糖核酸探针。 </li><li>执行在每豪根等人的 27协议原位杂交确认击倒如前所述11,20和在下面的代表性结果部分中讨论。 </li></ol></li><li>通过免疫组化确认击倒<ol><li>如果是可用的抗基因靶向的蛋白质产物，进行免疫组化分析，如前所述28，这有利于识别与击倒的表型分析20的最大水平的个体中例举的representati已经下文结果部分。 </li></ol></li></ol>

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E., 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=Evidence+of+RNAi+in+humans+from+systemically+administered+siRNA+via+targeted+nanoparticles.">Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles.</a> Nature. 464 (7921), 1067-1070 (2010).</li><li>Molinaro, R., 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=Polyethylenimine+and+chitosan+carriers+for+the+delivery+of+RNA+interference+effectors.">Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors.</a> Expert opinion on drug delivery. 10 (12), 1653-1668 (2013).</li><li>Singha, 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=Polymers+in+small-interfering+RNA+delivery.">Polymers in small-interfering RNA delivery.</a> Nucleic acid therapeutics. 21 (3), 133-148 (2011).</li></ol>

The authors have nothing to disclose.

脱乙酰壳多糖/干扰RNA纳米颗粒本文所描述的方法已被用于在A中幼虫发育期间有效地针对基因冈比亚 （ 图2,3）和A.埃及伊蚊 （ 图4，5，6，表1，表2）。壳聚糖的纳米颗粒可以与任一长的dsRNA或siRNA，这两者已被成功地用于在蚊子就证明了这里所描述的代表性的结果来制备。 dsRNA的合成比购买的siRNA更便宜，但更耗费人力。如果脱乙酰壳多糖/ siRNA的被使?...

在按蚊和Aedine属血液喂养蚊子传播负责几个人类的祸害最严重的致病因子。据估计，3.4十亿人的风险感染疟疾，负责每年全球有超过一半万人死亡。从感染疟疾结果由疟原虫属寄生虫，这是通过按蚊属感染的蚊子，包括主要的非洲病媒冈比亚按蚊 （叮咬传播给人类<a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> ，2014）1。 埃及伊蚊是登革热病毒，导致登革热，一种非特异性发热性疾病是世界上最普遍和显著虫媒病毒病的主要蚊子。登革热病毒是目前为&gt; 2.5十亿人在热带地区的威胁，与每年inciden每年24000人死亡导致大约〜50000000案件CE（ <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> ，2014）2。尽管蚊子传播的疾病，对人体健康的毁灭性的全球影响，预防和治疗这些疾病的有效手段缺乏。灭蚊是目前预防疾病的最佳方法。 用于控制通过媒介昆虫的遗传操作节肢动物传播的疾病的可能性已经认识超过四十年3。 A.转基因品系伊蚊设计有一个阻遏女性特有的飞型近期取得的潜力，利用转基因矢量控制策略成为现实4-6。这些进步都质疑研究人员确定了矢量控制和蚊子操纵基因功能的补充手段新的基因目标。基因表达ð维修uring发展，是女性飞蚊4的情况下，可能会促进新的矢量控制策略的阐释。但是，这主要是由于技术上的挑战，很少基因的功能已被鉴定A.发展过程中冈比亚，埃及伊蚊，或其他蚊种。 自从发现C.线虫 7，RNA干扰（RNAi），这是保守的动物，植物和微生物，已被广泛用于功能基因研究中的各种生物，包括昆虫8,9。 RNAi途径由Dicer酶，它劈开长​​双链RNA进入开始充当序列特异性RNA干扰短20-25个核苷酸长的siRNA。的siRNA沉默基因是在互补序列通过促 ​​进转录周转，裂解和翻译9的中断。长的dsRNA分子（通常为300-600碱基对）或定制的siRNA靶向particulař序列可用于在研究实验室用于沉默感兴趣的任何基因。当通过RNA干扰交付管理，研究人员可以控制时间，其中的基因沉默发起。因为它可以用来克服的挑战，如发育杀伤力或无菌，可阻碍生产和维护菌株轴承可遗传突变，一个昂贵的和劳动密集的过程，目前尚未在所有昆虫种类可用的这个优点是非常有用的。虽然基因沉默的RNA干扰的程度可以从基因而异基因，组织对组织和动物动物，RNAi技术被广泛地用于基因蚊子和其它昆虫8,9的功能分析。 三个干扰RNA递送策略已用于蚊子：显微注射，浸泡/局部施用，并摄取。进行了详细的历史和在昆虫中使用这三种技术中的比较，请参阅Yu 等人 8。 W¯¯E具有成功地使用显微注射10作为递送的siRNA的一种手段，以靶向A.发育基因伊蚊胚胎，幼虫，蛹和11-14。然而，这种劳动密集型的投放策略同时需要显微注射的设置和熟练的手。此外，显微注射是压力的生物体，一个混杂因素，特别是当行为表型进行评估。最后，显微注射递送不能扩展到田间进行矢量控制。作为替代，浸泡在干扰RNA溶液中的生物也成为一种流行的诱导基因沉默的手段，因为它是方便的，并且需要很少的设备或劳力。浸泡已经主要是在昆虫细胞系被应用研究8，但在最近的一项研究，在击倒A.达到伊蚊幼虫浸入dsRNA的15，其中动物似乎是摄取的溶液。然而，对于涉及多个experimenta的分析研究升组或表型，浸泡是相当昂贵的。洛佩兹-马丁内斯等人 16描述补液驱动的RNAi，一种新的方法来干扰RNA交付，涉及脱水生理盐水补液和用含有RNA干扰一滴水。这种方法并切断与整个动物浸没相关的成本，但是比注射更昂贵，并且可以将其应用到可容忍高渗透压物种的限制。此外，它是很难设想如何浸没或脱水/再水化浸渍方法可适于在磁场矢量控制。由于这些原因，用于后胚胎的研究，递送干扰RNA与摄入食物是一种可行的替代策略。 虽然摄入为基础的战略并不适用于所有的昆虫种类，也许最值得注意的是果蝇 ，口服给药干扰RNA与食物混合促进基因SIlencing在各种昆虫8,17，包括A.伊蚊成年人18。我们在A.描述壳聚糖纳米介导的RNA干扰冈比亚幼虫19，并已成功地应用这种方法为A.降低基因表达伊蚊幼虫20,21。这里，方法为这个RNAi的过程，这涉及截留通过聚合物壳聚糖干扰RNA的，是详尽。脱乙酰壳多糖/干扰RNA纳米颗粒通过聚阳离子的自组装通过氨基的脱乙酰壳多糖中的正电荷和由磷酸基团上的干扰RNA 19的骨干承载负电荷之间的静电引力干扰RNA形成。所述的程序是与两个长的dsRNA分子（以下简称为双链RNA）或双链的siRNA（以下简称为干扰）兼容。以下的合成，脱乙酰壳多糖/干扰RNA纳米颗粒混合，幼虫的食物，并传送到幼虫通过口服。这种方法相对便宜，只需要很少的设备和劳动力19，并有利于多种表型，包括行为20,21的分析的高通量分析。这种方法，可以适用于在其他昆虫，包括其它疾病的载体和昆虫农业害虫的基因沉默研究，有可能在各种其他动物物种的用于基因沉默。此外，脱乙酰壳多糖，一种廉价，无毒，可生物降解的聚合物22，有可能被利用在该领域为物种特异性蚊虫控制。

<table border="0" cellpadding="0" cellspacing="0" width="1429">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Reagent/Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:177px;">Catalog Number</td>
			<td style="width:663px;">Comments/Descriptions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR20</td>
			<td>for resuspension of interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR200</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-120</td>
			<td>for aliquoting resuspended interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR1000</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-046</td>
			<td>for preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Sodium Acetate, pH 4.5</td>
			<td>TEKnova</td>
			<td>S0299</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic Acid, AIRSTAR. ACS, USP, FCC Grade</td>
			<td>BDH</td>
			<td>VWR BDH3092-500MLP</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose Genetic Technology Grade</td>
			<td>MP Biomedicals LLC.</td>
			<td>800668</td>
			<td>to coat prepared interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf AG</td>
			<td>5415D</td>
			<td>to pellet interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, from shrimp shells</td>
			<td>Sigma-Aldrich</td>
			<td>C3646-25G</td>
			<td>to combine with interfering RNA for prepararation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Yeast</td>
			<td>Universal Food Corp</td>
			<td>NA</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-RNAi tool</td>
			<td>German Cancer Research Center</td>
			<td>NA</td>
			<td>for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goldfish food</td>
			<td>Wardley</td>
			<td>Goldfish FLAKE FOOD</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Heated water bath</td>
			<td>Thermo Scientific</td>
			<td>51221048</td>
			<td>to heat the interfering RNA/nanoparticles at 55oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>02-591-44</td>
			<td>for storage of ice used during the procedure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">liver powder</td>
			<td>MP Biomedicals LLC.</td>
			<td>900396</td>
			<td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microwave oven</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>to prepare 2% agarose solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petridish (100X15 mm)</td>
			<td>Fischer Scientific</td>
			<td>875713</td>
			<td>interfering RNA/nanoparticle feeding chamber for larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td>for preparation of buffers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blade</td>
			<td>Fischer Scientific</td>
			<td>12-640</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">siRNA</td>
			<td>Thermo Scientific/Dharmacon</td>
			<td>custom</td>
			<td>for preparation of siRNA/nanoparticles</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Sulfate, Anhydrous (Na2SO4)</td>
			<td>BDH/distributed by VWR</td>
			<td>VWR BDH0302-500G</td>
			<td>to prepare 50mM sodium sulfate solution in which the interfering RNA will be resuspended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermometer</td>
			<td>VWR</td>
			<td>61066-046</td>
			<td>to measure the water bath temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tooth picks</td>
			<td>VWR</td>
			<td>470146-908</td>
			<td>for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ultralow freezer</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>Fischer Scientific</td>
			<td>02-216-108</td>
			<td>for preparation of chitosan/interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Weight paper</td>
			<td>Fischer Scientific</td>
			<td>NC9798735</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
	</tbody>
</table>

chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae

蚊子造成更多人的痛苦比任何其他生物-和杀死每年有超过百万人。蚊子基因组计划推动研究蚊子生物学的新方面，包括在初级非洲疟蚊冈比亚按蚊和登革热和黄热病载体埃及伊蚊功能基因研究。核糖核酸干扰 - （RNAi-）介导的基因沉默已用于靶向目的基因在这两种疾病的载体蚊子物种。在这里，我们描述了一个程序，制备壳聚糖/干扰RNA纳米颗粒相结合，与食物和幼虫摄取。这种技术上简单，高通量，并且相对便宜的方法，这是与长双链RNA（dsRNA）的或小的干扰RNA（siRNA）分子相容，已经使用了若干在A中不同基因的成功击倒冈比亚和A.伊蚊幼虫。以下幼虫喂食，击倒，这是通过定量RT-PCR或原位杂交证实，可以持续至少通过后期蛹期。这种方法可以适用于各种各样的蚊子和其它昆虫物种，包括农业害虫，以及其它非模式生物。除了其在研究实验室工具，在未来，脱乙酰壳多糖，一种廉价，无毒和可生物降解的聚合物，有可能被利用在该领域。

<html> 冈比亚按蚊： 壳聚糖/双链RNA纳米颗粒由于脱乙酰壳多糖的氨基和dsRNA的磷酸基团之间的静电相互作用形成。 dsRNA的掺入效率为纳米粒子通常为90％以上，是因为通过从溶液耗尽dsRNA的测量。原子力显微镜图像表明，在直径壳聚糖的dsRNA粒径的平均值为140nm，从100-200纳米（ 图1）。 <img alt="图1"...

Watch this Scientific Journal Video about Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae at JoVE.com

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

这里，我们描述了通过使用脱乙酰壳多糖的抑制病媒蚊基因功能/干扰是由幼虫摄取的RNA纳米颗粒的过程。

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Research

JoVE Journal

Biology

14.2K 次观看.  Kansas State University. 这里，我们描述了通过使用脱乙酰壳多糖的抑制病媒蚊基因功能/干扰是由幼虫摄取的RNA纳米颗粒的过程。 

视频: Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

果蝇幼虫的欧诺细胞的解剖和脂滴染色

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...