Name: practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches
Uploaded: 2018-05-01T14:45:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

这项研究得到了台湾的赠款 (科学技术部, 多数 100-2923-002-002-MY3 和 106-2313-b-002-011-MY3 H.J.L.)、捷克共和国 (南波希米亚大学赠款机构、GAJU 赠款 065/2017/P y.h. L) 和西班牙 (西班牙经济和竞争力部, 赠款 CGL2012-36251 和 CGL2015-64727-P 王啸波和加泰罗尼亚政府, 赠款 2014 SGR 619 王啸波);它还得到欧洲经济和区域发展基金 (菲德基金王啸波) 的财政支助。

1. dsRNA 的合成和制备
<ol><li>确定目标基因 3 ' 未翻译区域中的 dsRNA 目标点。dsTub 用于靶向α蛋白 (盆) 基因 (基因库加入号: KX228233), dsEGFP 作为负 dsRNA 控制是设计从序列的增强绿色荧光蛋白 (EGFP;基因库加入号码: LC311024)。</li>
	<li>进行标准 PCR 扩增, 合成 dsRNA 模板与基因特异引物包含 T7 启动程序序列 (5 '-TAATACGACTCACTATAGGG-3 ')。PCR 扩增条件和引物信息是由林et报告的。(2017)12 (请参见材料表)。</li>
	<li>按照制造商的说明, 使用 T7 转录套件 (参见材料表) 进行 dsRNA 准备。 注意:要产生高数量的 dsRNA, 混合两个反应在一个管 (总共40µL), 并孵化在37°c 过夜。</li>
	<li>将2µL 的 DNase (参见材料表) 添加到 dsRNA 样本中, 15 分钟37摄氏度, 以消化 DNA 模板。</li>
	<li>添加200µL 萃取试剂 (参见材料表) 和40µL 氯仿, 然后是涡流。</li>
	<li>离心机为10分钟在 1.3万 x g 在4°c, 然后收集上清 (150 µL) 在一个新的离心管。</li>
	<li>在冰上加入150µL 异丙醇并孵化15分钟, 沉淀 dsRNA。</li>
	<li>离心机15分钟在 1.3万 x g 在4°c, 然后去除上清。</li>
	<li>添加200µL 70% 乙醇, 以洗涤 dsRNA 丸。离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>除去乙醇, 重复乙醇洗涤步骤。</li>
	<li>用离心真空浓缩器去除乙醇和干 dsRNA 颗粒3分钟, 然后并用重悬 dsRNA 无 RNase 水。</li>
	<li>用微体积紫外-可见光分光光度计 (参见材料表) 确定纯化 dsRNA 的数量, 并根据制造商的说明调整到所需浓度 (例如, 0.25 µg/µL, 以准备dsRNA lipoplexes)。</li>
	<li>将 dsRNA 样品存放在摄氏-20 摄氏度, 长达3月。</li>
</ol>2. dsRNA Lipoplexes 的制备
<ol><li>通过添加4µL 5% 葡萄糖溶液稀释4µL 的转染试剂 (参见材料表)。通过添加4µL 5% 葡萄糖溶液稀释4µL 的 dsRNA (1 µg)。</li>
	<li>立即将稀释脂质体试剂溶液加入稀释后的 dsRNA 溶液中。漩涡简单地混合, 然后孵育15分钟在25摄氏度。 注意:不要以相反的顺序混合解决方案。</li>
	<li>dsRNA lipoplexes 已经准备好使用了。使用1小时内准备, 并在1小时后丢弃。 注意:根据制造商的指示, 应尽快使用 lipoplexes。</li>
</ol>3. 从血淋巴和中肠果汁中收集胞外酶
<ol><li>
昆虫盐水缓冲液 (10x 库存)
	<ol>
<li>混合900毫升双蒸馏水, 109.3 克氯化钠, 15.7 克氯化钾, 6.3 克 CaCl2, 8.3 g 氯化镁2· 6H2O。</li>
		<li>将 pH 值调整为 6.8, 并填充至1000毫升。</li>
	</ol>
</li>
	<li>
血淋巴收集
	<ol>
<li>麻醉在冰上的蟑螂, 直到他们不移动3分钟。</li>
		<li>用两个手指 (拇指和食指) 抓住蟑螂, 把蟑螂的腹侧打开。</li>
		<li>使用解剖剪刀切断后腿髋部的尖端。 注意:切断髋和转子之间的连接交叉口。另一部分的髋部切除术对收集血淋巴的效率较低。</li>
		<li>用中指轻轻挤压腹部, 用10µL 微从切口收集血淋巴出血。</li>
		<li>从几个蟑螂 (5 个人) 的血淋巴池中离心管。 注意:把离心管放在冰上以防黑化。从一只雄性蟑螂身上获得的血淋巴平均容积为 2-3 µL。</li>
		<li>十年代用台式离心机简单地旋转血淋巴。</li>
		<li>转移所需血淋巴量 (即, 10 µL), 并将其与50µL 1x 昆虫盐水缓冲液混合。</li>
		<li>离心机为10分钟在 1000 x g 在4°c 下包囊。将上清液转移到干净的离心管上。</li>
		<li>通过测量 A28015, 用微体积紫外-可见光分光光度计定量测定总蛋白浓度。</li>
		<li>调整每个样品具有相同的蛋白质浓度 (6 毫克/µL 的总蛋白)。</li>
	</ol>
</li>
	<li>
中肠果汁系列
	<ol>
<li>麻醉把蟑螂放在冰上。用昆虫引脚将蟑螂腹侧用冷1x 昆虫盐缓冲器固定在解剖板上。</li>
		<li>用细镊子解剖腹部。将整个肠道 (包括前, 中, 后肠道) 移到新鲜的盘子里, 1x 昆虫盐缓冲。</li>
		<li>取出前肠和后肠道, 迅速将中肠转移至含有100µL 昆虫盐水缓冲液的离心管。</li>
		<li>midguts 从六只蟑螂在同一管, 和漩涡十年代。</li>
		<li>离心机为10分钟, 在 1000 x g 在4°c, 包囊和肠道组织的旋转。将上清液转移到干净的离心管上。</li>
		<li>通过测量 A28015, 用微体积紫外-可见光分光光度计定量测定总蛋白浓度。</li>
		<li>调整每个样品具有相同的蛋白质浓度 (6 毫克/µL 的总蛋白)。</li>
	</ol>
</li>
</ol>4. dsRNA 降解试验
<ol><li>新鲜地准备4µL 赤裸 dsRNA 或 dsRNA lipoplexes (1 µg)。</li>
	<li>混合 dsRNA 溶液与10µL 的昆虫盐水缓冲 (控制), 提取酶的血淋巴, 或中肠果汁。</li>
	<li>添加2µL EGTA (20 毫米) 或无 RNase 水, 以分离样品作为控制酶活性的抑制。</li>
	<li>孵育1小时或更长 (6, 12, 或24小时) 在25°c。</li>
	<li>加入200µL 萃取试剂和40µL 氯仿, 然后涡流。</li>
	<li>离心机为10分钟在 1.3万 x g 在4°c, 然后收集上清 (150 µL) 在一个新的离心管。</li>
	<li>在冰上加入150µL 异丙醇并孵化15分钟, 沉淀 dsRNA。</li>
	<li>离心机15分钟在 1.3万 x g 在4°c, 然后去除上清。</li>
	<li>添加200µL 70% 乙醇, 以洗涤 dsRNA 丸。离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>除去乙醇, 重复乙醇洗涤步骤。</li>
	<li>用离心真空浓缩器将乙醇和干 dsRNA 颗粒去除3分钟并用重悬 dsRNA, 10 µL 无 RNase 水。</li>
	<li>在1.5% 琼脂糖凝胶上通过凝胶电泳检查 dsRNA 的完整性。</li>
</ol>5. dsRNA 的口服管理
<ol><li>在广告随意饮食中保持蟑螂 (狗周)。在 dsRNA 摄入实验前一天, 从蟑螂那里夺走了水的供应。</li>
	<li>用挠性钳抓住它的翅膀 (图 1,步骤 5) 来抓蟑螂。 注意:不要抓蟑螂的身体部位。</li>
	<li>准备 dsRNA lipoplexes, 以及裸 dsRNA, 以供喂养, 如第2节所述。 注意:dsRNA lipoplexes 应在进食前准备新鲜。</li>
	<li>饲料4µL dsRNA lipoplexes 或裸 dsRNA (250 吴) 与微。</li>
	<li>慢慢地吸管 dsRNA 溶液的水滴靠近蟑螂的口器, 让蟑螂完全摄取水滴。</li>
	<li>每天进行两次摄取实验 (1 小时后灯亮起, 1 小时熄灯前) 持续8天或16天。 注意:所有蟑螂获得水只有通过手动喂养 dsRNA 溶液或控制试剂 (例如, 核酸酶免费水, 葡萄糖溶液, 脂质体试剂) 在摄取实验。</li>
	<li>在16天的 dsRNA 实验后, 给蟑螂提供水瓶。</li>
</ol>6. 评估击倒
<ol><li>在选择的时间点 (2, 9, 17 天后 dsRNA 摄取化验), 收集 dsEGFP 和 dsTub 处理的昆虫和解剖所选择的组织 (例如, 中肠)。</li>
	<li>进行定量实时 pcr (qRT pcr)。qRT-PCR 扩增条件和引物信息的报告, 林et al。(2017)12。</li>
	<li>每天评估控制和 dsRNA 治疗的昆虫, 以检查死亡率和清除不再移动的蟑螂。</li>
</ol>

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作者没有什么可透露的。

本协议提出了一种有效的 rna 干扰方法, 通过口服 dsRNA lipoplexes, 包括防止核糖核酸消化在中肠果汁的德国蟑螂。正如在其他昆虫种类的研究中所显示的, 通过口服 dsRNA 的干扰效应很差, 主要是由于 dsRNA8,9,10的退化。该协议产生脂质体, 作为保护性的工具, 在口头交付 dsRNA 抗降解的肠道。此外, dsRNA lipoplexes 的制备方法简单, 易于应...

rna 干扰已被证明是一种有效的方法来击倒基因表达通过一个机制的转录后沉默途径触发的 dsRNA 分子在许多真核生物1。在过去的十年中, rna 干扰已经成为一个有用的工具来研究基因从发展到行为的功能, 通过消耗特定基因的表达, 通过注射和/或喂养 dsRNA 在昆虫的各种分类器2,3。由于损耗效应的特殊性和鲁棒性, 目前正在将 rna 干扰的应用视为害虫控制管理的潜在策略4,5。然而, 在昆虫种类之间, rna 干扰的效率差别很大, 这取决于不同基因的靶向和传递方法。越来越多的证据表明, 由 ribonucleases 降解的 dsRNA 的不稳定性是 rna 干扰5、6的有限功效的一个关键因素。例如, Manduca 天蛾中的低干扰敏感度被解释为: 与血淋巴混合的 dsRNA 在1小时的7中迅速退化。同样, 中肠碱性核酸的存在, 有效地降低了摄取 dsRNA, 在不同的昆虫顺序中与低 rna 干扰效率有很强的相关性8,9,10。
dsRNA 的口头交付对于在害虫防治战略中应用 rna 干扰特别有趣, 但尚未开发出一种延缓中肠核酸降解 dsRNA 的方法, 这将有可能确保有效通过喂食进行 rna 干扰。然而, dsRNA 的口服的对停止响应是通过喂养大量的 dsRNA ,例如50µg在家蚕, 或连续喂养8天 (总共8µg dsRNA) 的蝗虫物种。德国蟑螂,德国 germinica, 对通过 dsRNA11,12,13,14的注入而对 rna 的干扰非常敏感, 但对 dsRNA 通过喂食没有响应。最近, 林等。(2017) 证明了脂质体载体封装的 dsRNA 成功地进行了 rna 干扰, 以击倒中肠中的α蛋白基因表达, 并触发德国蟑螂15的显著死亡率。由于中肠 dsRNA 的降解是口服 rna 干扰的限制因素, 脂质体载体作为一种保护 dsRNA 免受降解的载体, 在肠道内具有较强核酸酶活性的其他昆虫中很容易被应用。值得注意的是, 选择特定转染试剂的原因 (参见材料表) 我们作为脂质体载体在目前的协议是, 它已被测试的昆虫细胞线转染较少的毒性, 根据制造商的说明。通过对不同脂质体转染系统的比较 Gharavi et 。(2013)16中, 转染小干扰 RNA (siRNA) 的效率大约与其他昆虫17、18中用于 dsRNA 传送系统的其他商用系统之间的效果大致相同..此外, 我们的喂养方法是足够小心, 以确保每只蟑螂摄取适量的 dsRNA, 结果是稳健的和确认的。总之, 本协议和结果表明, 使用 dsRNA lipoplexes 提高了 dsRNA 的稳定性, 为 rna 干扰的策略设计开辟了大门, 这是今后害虫防治的一个有前途的途径。

<table><tbody><tr><td>GenJe Plus DNA in vitro Transfection reagent</td><td>SignaGen</td><td>SL100499</td><td>for lipoplexes preparation</td></tr><tr><td>Blend Taq plus</td><td>TOYOBO&amp;nbsp;</td><td>BTQ-201</td><td>for PCR</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ABI&amp;nbsp;</td><td>4385612</td><td>for qPCR</td></tr><tr><td>FirstChoice RLM-RACE Kit</td><td>Invitrogen</td><td>AM1700</td><td>for 3&#039; UTR identification&amp;nbsp;</td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Invitrogen</td><td>AMB13345</td><td>for dsRNA synthesis</td></tr><tr><td>TURBO DNase</td><td>Invitrogen</td><td>AM2239</td><td>remove DNA template from dsRNA</td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>15596018</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>RQ1 RNase-Free Dnase</td><td>Promega</td><td>M6101</td><td>remove DNA template from total RNA&amp;nbsp;</td></tr><tr><td>chloroform&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C2432</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>I9516</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>ethanol</td><td>Sigma-Aldrich</td><td>24102</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Diethyl pyrocarbonate, DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>for RNase free water preparation</td></tr><tr><td>glucose solution</td><td>Sigma-Aldrich</td><td>G3285</td><td>for lipoplexes preparation</td></tr><tr><td>Sodium chloride, NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>insect saline buffer formula</td></tr><tr><td>Potassium chloride, KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>insect saline buffer formula</td></tr><tr><td>Calcium chloride, CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1016</td><td>insect saline buffer formula</td></tr><tr><td>Magnesium chloride hexahydrate, MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670</td><td>insect saline buffer formula</td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E3889</td><td>enzyme inhibitor&amp;nbsp;</td></tr><tr><td>dissecting scissor</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>fine tweezers</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>flexible tweezer</td><td>F.S.T.</td><td></td><td>cockroach holding&amp;nbsp;</td></tr><tr><td>pipetman</td><td>RAININ</td><td>P10</td><td>sample preparation</td></tr><tr><td>microcentrifuge tube</td><td>Axygen</td><td>MCT175C, PCR02C</td><td>sample preparation</td></tr><tr><td>pipette tip&amp;nbsp;</td><td>Axygen</td><td></td><td>sample preparation</td></tr><tr><td>vortexter</td><td>Digisystem</td><td>vm1000</td><td>sample preparation</td></tr><tr><td>Minispin centrifuge</td><td>The Gruffin Group</td><td>GMC 206</td><td>for liquid spin down&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>ALC</td><td>PK121R</td><td>sample preparation</td></tr><tr><td>pH meter&amp;nbsp;</td><td>JENCO</td><td>6071</td><td>for pH adjust</td></tr><tr><td>micro-volume spectrophotometer</td><td>Quawell</td><td>Q3000</td><td>nucleic acid quantitative</td></tr><tr><td>PCR Thermal cycler</td><td>ABI&amp;nbsp;</td><td>2720</td><td>for template PCR or&amp;nbsp; dsRNA synthesis incubation&amp;nbsp;</td></tr><tr><td>quantitative real-time PCR</td><td>ABI&amp;nbsp;</td><td>StepOne plus</td><td>gene expression quantitative</td></tr><tr><td>Centrifugal Vacuum Concentrators</td><td>eppendorf</td><td>5301</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Multipette&amp;nbsp;</td><td>eppendorf</td><td>xstream</td><td>for real-time PCR sample loading&amp;nbsp;</td></tr><tr><td>Agarose I</td><td>amresco</td><td>0710</td><td>for nucleic acid electrophoresis</td></tr><tr><td>tub gene specfifc forward preimer</td><td>tri-I biotech</td><td></td><td>GGG ACA AGC CGG AGT GCA GA</td></tr><tr><td>tub gene specfifc reverse preimer</td><td>tri-I biotech</td><td></td><td>TCC TGC TCC TGT CTC GCT GA</td></tr><tr><td>dsTub template forward primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&amp;nbsp;</td></tr><tr><td>dsTub template reverse primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&amp;nbsp;</td></tr><tr><td>dsEGFP template forward preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td></tr><tr><td>dsEGFP template reverse preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td></tr><tr><td>tub qPCR forward primer</td><td>tri-I biotech</td><td></td><td>GGA CCG CAT CAG GAA ACT GGC</td></tr><tr><td>tub qPCR reverse preimer</td><td>tri-I biotech</td><td></td><td>CCA CAG ACA GCC TCT CCA TGA GC</td></tr><tr><td>ef1 qPCR forward primer</td><td>tri-I biotech</td><td></td><td>CGC TTG AGG AAA TCA AGA AGG A</td></tr><tr><td>ef1 qPCRreverse preimer</td><td>tri-I biotech</td><td></td><td>CCT GCA GAG GAA GAC GAA G</td></tr></tbody></table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

rna 干扰在揭示众多基因的生物学功能方面得到了广泛的应用, 并被设想为一种害虫控制工具, 其主要基因表达被打乱。虽然报告了不同的方法, 如注射, 喂养和浸泡, 成功地交付双链 RNA (dsRNA), 通过口服 dsRNA 的 rna 干扰的效率在不同的昆虫群中是高度可变的。德国蟑螂, 德国的大蠊, 对 dsRNA 的注射高度敏感, 正如此前发表的许多研究显示的那样。本研究描述了用脂质体载体包裹 dsRNA 的方法, 足以延缓中肠果汁对 dsRNA 的降解。值得注意的是, 脂质体包覆的 dsRNA 的连续喂养大大降低了中肠蛋白的表达, 导致了蟑螂的死亡。总之, dsRNA lipoplexes 的配制和利用, 保护 dsRNA 免受核酸的危害, 是今后害虫防治的一种实用方法。

在图 1中介绍了 dsRNA 口头交付协议的简化方案, 其中显示了准备 dsRNA lipoplexes 的关键步骤。
为了研究脂质体载体对 dsRNA 降解的保护作用, 对B...................dsTub lipoplexes 在中肠果汁中进行了一种前体检测, dsRNA随后对1.5% 琼脂糖凝胶进行了分析。图 2显示了在B. 小蠊...

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

本手稿通过口服双链 RNA 包裹在脂质体中, 证明了德国蟑螂中肠内基因表达的耗竭。

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

practical-use-rna-interference-oral-delivery-double-stranded-rna

Research

JoVE Journal

Biology

9.3K 次观看.  National Taiwan University. 本手稿通过口服双链 RNA 包裹在脂质体中, 证明了德国蟑螂中肠内基因表达的耗竭。

视频: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

科研

遗传学

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

环境科学

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

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 ...

发育生物学

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

生物学

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

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 ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

<meta charset="utf8"></head><body>纳米颗粒介导的 dsRNA 喂养用于家蚕幼虫基因沉默

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.

<meta charset="utf8"></head><body>作者聚焦：通过饲喂壳聚糖纳米颗粒推进 dsRNA 递送家蚕的基因沉默研究

Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对 家蚕 Bombyx 的 干扰

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of ...

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

摘要

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此视频中的章节

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA干扰在美洲蟑螂中的应用

RNAi的干扰双链RNA注射到果蝇胚胎

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对家蚕 Bombyx 的干扰

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

摘要

探索更多视频

此视频中的章节

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA干扰在美洲蟑螂中的应用

RNAi的干扰双链RNA注射到果蝇胚胎

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对 家蚕 Bombyx 的 干扰

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对家蚕 Bombyx 的干扰