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.蚊種と飼育<ol><li> A.を維持ハマダラカ G3 とA.ネッタイシマカリバプールIB12株（下の代表的研究に使用される）または関心のある他の株の標準実験室の練習にまたは以前に23,24説明したように従った。 </li></ol> 2. dsRNAおよびsiRNAの設計と製造<ol><li>設計プライマーは、関心対象の遺伝子に特異的な長いdsRNAテンプレートを構築した。 E-のRNAiツール25を使用してください。として選択または方法に従ってのdsRNA産生は、以前に19を記載した。 <ol><li>陰性対照のために、緑色蛍光タンパク質（GFP）、β-ガラクトシダーゼ（β-gal）、または蚊では発現されないいくつかの他の遺伝子の配列に対応するdsRNAが19を合成する。所望であれば、以下に要約する代表的な結果を生成するために利用dsRNAは19を陽性対照として使用することができる。ストアdsRNAは、-80℃で無RNAse水に溶解; C必要になるまで。 </li></ol></li><li>あるいは、設計遺伝子特異的siRNA標準ラボ慣行または以前に記載10のようによれ。すべての蚊の遺伝子に対応していないネガティブコントロールsiRNAを設計するためにノックダウンsiRNAの配列をスクランブル。評判の良いベンダーの数を介して使用できますカスタムsiRNAを、購入してください。 <ol><li>所望であれば、以下に要約する代表的な結果を得るために用いたsiRNA 20,21は、陽性対照として使用することができる。必要になるまでストアsiRNAは、-80℃でRNaseフリー水に溶解した。 </li></ol></li></ol> 3. R​​NAナノ粒子干渉/キトサンの準備<ol><li>既製のRT 100mMの硫酸ナトリウムを集める（脱イオンH 2 O中の100mMのNa 2 SO 4）、0.1 M酢酸ナトリウム（脱イオンH 2 O中の0.1 M NAC 2 H 3 O 2 -0.1 M酢酸、pH4.5）中バッファ。 </li><li>キトサンを溶解する（≥75％脱アセチル化された）は、0.1 M酢酸ナトリウム緩衝液（w / v）の希釈標準溶液、0.02％を作るために、使用前にRTで溶液を維持する。 </li><li> H 2 Oの脱イオン50μl中のdsRNAまたはsiRNAを溶解し、50 mMの硫酸ナトリウム100μlのあたりのdsRNA / siRNAの32μgの100μlの溶液を作製するために100 mMの硫酸ナトリウムバッファーに追加します。 </li><li>のdsRNA / siRNA溶液にキトサン溶液100μlを加え、その後、1分間55℃の水浴中で混合物を加熱する。キトサン溶液の100μlに50 mMの硫酸ナトリウム100μlを加えることで制御を設定し、同じ手順に従います。 </li><li>ナノ粒子の形成を容易にするために、室温で、高速ボルテックスで30秒間、直ちに溶液を混合する。 </li><li>遠心分離し、ペレットを表示するか、その時間後室温で10分間、13,000×gで混合。新しい1.5 mlチューブに上清を移し。蚊の食品を調製するために使用する前に室温で≈10分間、ペレットを空気乾燥。 </li><li> M上清に残ったのdsRNA / siRNAの総量を計算するために、ブランクとしてコントロール（3.5を参照）からの上清を使用して上清中のdsRNA / siRNAの濃度をeasure。のdsRNA / siRNAの出発量の差を使用して、ナノ粒子中に封入さのdsRNA / siRNAの割合を計算するために、上清中に残存する量。この充填効率は、通常90％以上である。 </li><li>以上のナノ粒子が必要な場合は、同じ手順を繰り返します。すぐに乾燥したナノ粒子を使用してください。使用前に粒子の低温貯蔵の影響が評価されていない。 </li></ol> 4.モスキート食品は、RNAナノ粒子干渉/キトサンを含む準備<ol><li> （w / v）の脱イオン水に、アガロースを溶融し、2％アガロース溶液1mlを調製し、使用前に55℃の水浴中で融解したアガロース溶液を保つ。 </li><li>魚の餌フレークを混ぜ（47％粗タンパク質、分。粗脂肪10％、最大粗繊維3％）で、乾燥酵母2：1の比（w / w）である。 （第50号米国標準試験ふるいを通過できる）を乳鉢と乳棒で小さな粒子に混合物を挽く。どちらかすぐに、色が茶色がかったあるべき地上の食べ物を、使用するか、または数週間4℃で密封容器に保管してください。 </li><li> 1.5ミリリットルチューブでは、つまようじで3.7節からの乾燥ナノ粒子で地面食品の6 mgのミックス。 </li><li>食品·ナノ粒子混合物に2％のプレ溶融したアガロースゲル溶液の30μlのを追加します。つまようじまたはピペットチップを使用してすぐに、徹底的にかき混ぜる。 </li><li>すぐに蚊の幼虫を養うために食品やナノ粒子を含むゲルを使用してください。ゲルが完全に室温で固化されると代替的に、4℃でゲルを格納し、使用する次の日に、または後で使用するために-80℃で。 </li></ol>食べ物は、RNAナノ粒子干渉/キトサンを含有する蚊の幼虫を餌5. <ol><li>給餌A.ハマダラカ蚊の幼虫： <ol><li> Siのを削除爪楊枝を使用して1.5ミリリットルチューブからngleゲルペレットときれいなカミソリの刃や歯のピックを使用して6等しいスライスにカット。 </li><li>脱イオン水100mlを含む500mlペトリ皿に20三齢幼虫を転送します。 </li><li> 4日間、一日一回ペトリ皿当たりゲルペレット（細かく小さな断片に切り刻ま）の1スライスを追加することによって蚊の幼虫フィード。大幅にサイズが縮小または翌日までに完全に存在する必要があり、ペレット、上の幼虫の摂食を必ず守ってください。時間が経過すると、蚊が遅れて4齢幼虫に発展する。 </li><li>実験中の目に見える表現型の変化を記録します。 4日間の期間の終了時にセクション6で説明したように転写レベルおよび他の表現型の変化を調べます。 </li></ol></li><li>給餌A.ネッタイシマカの蚊の幼虫： <ol><li>清潔なカミソリの刃や爪楊枝を使って6等しいスライスにゲルペレットをカット。 </li><li>卵孵化Fiの後に50歳の同期24時間を置きます≈40mlの脱イオン水中のシャーレに1の幼虫。 </li><li>その日の残りの1グラウンド魚の餌フレークとドライイースト：その後2の正規の幼虫の食事に戻って幼虫を転送する、4時間/日のペトリ皿当たりの幼虫1スライスを養う。 4幼虫齢を通して毎日の手順を繰り返します。 </li><li>希望発達の時点でセクション6で説明したように転写レベルおよび他の表現型の変化を調べます。 </li></ol></li></ol>遺伝子ノックダウンの6.確認<ol><li> Aの定量RT-PCRによる相対転写産物の定量化ネッタイシマカとA.ハマダラカの幼虫。 <ol><li>少なくとも10の3つの生物学的複製とを実行し、分析のqRT-PCRアッセイは11,19,20が記載されている実験の幼虫対コントロールをプールし、または標準的な実験手順に従って。代表的な結果を以下に含まれています。 </li><li>特定の組織タイプ/本体部分（ すなわち 、脳またはアンテナ）の分析のために、perfo目的の組織を回復する解剖以下6.1.1に記載のように定量RT-PCRのrm（例えば、マイソールら 21を参照されたい）。 </li></ol></li><li> in situハイブリダイゼーションによるノックダウンの確認<ol><li>ジゴキシゲニン標識アンチを合成し、標準的な実験室の練習にまたは26に記載のように応じて制御リボプローブを感知。 </li><li> 11,20以前に説明し、以下の代表的な結果のセクションで説明したようにノックダウンの確認のためハウゲンら 27プロトコルごとin situハイブリダイゼーションを実行します。 </li></ol></li><li>免疫組織化学によるノックダウンの確認<ol><li>標的遺伝子のタンパク質産物に対する抗体が利用可能である場合、以前representatiに例示されるように表現型分析20のためのノックダウンの最大レベルを有する個体の同定を容易に28を 、説明したように、免疫組織化学を行う下記の結果セクションVEの。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+with+polyvalent+siRNA-nanoparticle+conjugates.">Gene regulation with polyvalent siRNA-nanoparticle conjugates.</a> Journal of the American Chemical Society. 131 (6), 2072-2073 (2009).</li><li>Davis, M. 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.

本明細書に記載のキトサン/干渉RNAナノ粒子法は、効果的にAの発育中の遺伝子を標的とするために使用されているハマダラカ （ 図2、図3）およびA.ネッタイシマカ （ 図4、図5、図6、表1、表2）。キトサンナノ粒子は、本明細書に記載の代表的な結果によって証明されるように蚊に成功裡に使用されている両方とも、長いdsRNAまたはsiRNAのいずれか?...

ハマダラカとAedine属の血液供給の媒介蚊は、人類の最悪の災いのいくつかの責任病気の原因となる物質を伝達する。推定34億の人々が毎年世界中でオーバーワン50万人の死亡の原因であるマラリアを、縮小するための危険にさらされている。 マラリア原虫属による感染からマラリアの結果。主要なアフリカのベクトルハマダラカ （含むハマダラカ属の感染した蚊の刺さを通じて人々に送信され、寄生虫、 <a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> 、2014）1。 ネッタイシマカは、デング熱、世界で最も広範かつ重要なアルボウイルス疾患である非特異的な熱性疾患の原因となるデング熱ウイルス、の主要な蚊ベクトルである。デング熱ウイルスは毎年incidenで、現在熱帯&gt; 25億人への脅威である毎年〜24000人が死亡（結果として約5000万例のCE <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> 、2014）2。人の健康に蚊が媒介する病気の壊滅的な世界的な影響にもかかわらず、これらの疾患の予防及び治療の効果的な手段が不足している。モスキートコントロールは、現在、病気の予防の最善の方法です。 ベクトル昆虫の遺伝子操作によって節足動物媒介疾患を制御するための可能性は40年以上3認識されてきた。 A.のトランスジェニック系統抑制性女性特有飛べ表現型を有するように操作ネッタイシマカは、最近、トランスジェニックベクターコントロール戦略現実4-6を使用するための可能性を作った。これらの進歩は、ベクター対照と媒介蚊で遺伝子機能を操作するさらなる手段のための新規の遺伝子標的を同定するために研究者が挑戦してきた。遺伝子発現dの変質を雌飛べ蚊4でそうであったようにuring開発は、新規ベクトル制御戦略の解明を促進することができる。しかし、主として技術的な課題に、非常に少数の遺伝子の機能は、Aの開発中に特徴付けられているハマダラカ、ネッタイシマカ、または他の蚊の種。 C.年の発見以来エレガンス 7、動物、植物および微生物において保存されているRNA干渉（RNAi）は、広範囲の昆虫8,9を含む多種多様な生物において機能的な遺伝学的研究のために使用されてきた。 RNAi経路は、配列特異的干渉RNAとして機能する短い20-25ヌクレオチド長のsiRNAに切断する長いdsRNAダイサーによって開始される。転写産物のターンオーバー、切断、および翻訳9の破壊を促進することにより、配列が相補的であるsiRNAは沈黙遺伝子。 particulaをターゲットに長いdsRNA分子（通常は300〜600塩基対）、またはカスタムのsiRNArの配列は、目的の任意の遺伝子をサイレンシングするために研究室で使用することができる。干渉RNAが送達されたときに管理することにより、研究者は、遺伝子がサイレンシングを開始する時間を制御することができる。それは遺伝変異を有する株の生産やメンテナンス、すべての昆虫種ではまだ使用できません高価で労働集約的なプロセスを妨げることができるように、発達致死や無菌性などの課題を克服するために使用することができようにこの利点に便利です。 RNAiによる遺伝子サイレンシングの程度は、動物への遺伝子、組織、組織、および動物に遺伝子変化し得るが、RNAiは広く蚊や他の昆虫8,9における遺伝子の機能解析のために使用される。 三つの干渉RNA送達戦略は、蚊に使用されてきた：微量注入、浸漬/局所適用、経口摂取を。詳細な歴史と昆虫におけるこれらの3つの技術の利用の比較のために、ゆうら 8を参照してください。 Weは成功してAの発生遺伝子をターゲットとするsiRNAを送達する手段としてマイクロインジェクション10を使用していたネッタイシマカの胚、幼虫、蛹11-14。しかし、この労働集約的な配信戦略は、マイクロインジェクションのセットアップと熟練した手の両方を必要とします。また、マイクロインジェクションは、行動の表現型が評価される場合は特に、生物、交絡因子にストレスが多い。最後に、マイクロインジェクション送達は、ベクトル制御のためのフィールドを拡張することができない。それは便利であり、ほとんどの機器や労力を必要とする別の方法として、RNA溶液を干渉する生物を浸すことはまた、遺伝子サイレンシングを誘導する人気の手段となっている。浸漬は、主に昆虫細胞株に適用された8を研究が、最近の研究では、ノックダウンA.で達成されたネッタイシマカの幼虫は、動物が摂取するように思われたdsRNA 15の溶液に浸漬した。ただし、複数のエクスペリの分析を含む研究のためのL基または表現型は、浸漬はかなり高価である。ロペス·マルティネスら 16は、RNAi、干渉RNAを含む水の一滴と食塩水と水分補給で脱水を伴うRNAの配信を妨害するための新規のアプローチを駆動水分補給を説明した。このアプローチは、動物全体の浸漬に関連するコストを削減するが、マイクロインジェクションよりも高価であり、高い浸透圧に耐えることができる化学種への適用に限定されてもよいしない。また、浸漬、脱水/再水和の浸漬方法は、フィールド内のベクトル制御に適合させることができるか想像することは困難である。これらの理由から、後胚の研究のために、摂取した食物との干渉RNAの送達は実行可能な代替戦略です。 摂取ベースの戦略は、すべての昆虫種では動作しませんが、おそらく最も顕著キイロショウジョウバエ 、食品と混合したRNA干渉の経口送達は、遺伝子のSiを推進してきましたA.含む昆虫8,17、さまざまなlencing ネッタイシマカ大人18。私たちは、Aにキトサンナノ粒子媒介RNAiを説明したハマダラカ幼虫 19に成功A.における遺伝子発現の低減のためのこのアプローチを適用しているネッタイシマカの幼虫20,21。ここで、高分子キトサンによるRNA干渉の包括を伴うこのRNAiの手順、方法論は、詳細です。キトサン/干渉RNAナノ粒子は、キトサン中のアミノ基の正電荷とRNA 19干渉の主鎖にリン酸基により担持された負電荷間の静電力を介して干渉RNAとポリカチオンの自己集合により形成される。記載された手順は、長いdsRNA分子（以下のdsRNAとも呼ばれる）または二本鎖siRNA（以下のsiRNAと呼ばれる）の両方と互換性がある。以下の合成、キトサン/ RNAナノ粒子が幼虫食品と混合に配信される干渉経口摂取を通じて幼虫。この方法は、比較的安価であり、少ない設備および労力19を必要とし、行動20,21の解析を含む複数の表現型のハイスループット分析を容易にする。他の疾患ベクターおよび昆虫農業害虫を含む他の昆虫の遺伝子サイレンシング研究のために適合させることができるこの方法は、潜在的に他の種々の動物種における遺伝子サイレンシングのために使用することができる。また、キトサン、安価な、非毒性および生分解性ポリマー22は 、潜在的に種特異的な蚊の駆除のための分野において利用することができる。

<table><tbody><tr><td>0.02 ml pipetteman</td><td>Rainin</td><td>PR20</td><td>for resuspension of interfering RNA</td></tr><tr><td>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><td>0.5 ml graduated tube&amp;nbsp;</td><td>Fischer Scientific</td><td>05-408-120</td><td>for aliquoting resuspended interfering RNA</td></tr><tr><td>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><td>1.5 ml graduated tube&amp;nbsp;</td><td>Fischer Scientific</td><td>05-408-046</td><td>for preparation of interfering RNA/nanoparticles</td></tr><tr><td>1M Sodium Acetate, pH 4.5</td><td>TEKnova</td><td>S0299</td><td>to prepare sodium acetate buffer</td></tr><tr><td>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><td>Agarose Genetic Technology Grade</td><td>MP Biomedicals LLC.</td><td>800668</td><td>to coat prepared interfering RNA/nanoparticles</td></tr><tr><td>Centrifuge</td><td>Eppendorf AG</td><td>5415D</td><td>to pellet interfering RNA/nanoparticles</td></tr><tr><td>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><td>Dry Yeast</td><td>Universal Food Corp</td><td>NA</td><td>to prepare mosquito larval food with interfering RNA/nanoparticles</td></tr><tr><td>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><td>goldfish food</td><td>Wardley</td><td>Goldfish FLAKE FOOD</td><td>to prepare mosquito larval food with interfering RNA/nanoparticles</td></tr><tr><td>Heated water bath</td><td>Thermo Scientific</td><td>51221048</td><td>to heat the interfering RNA/nanoparticles at 55&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Ice</td><td>not applicable</td><td>not applicable</td><td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Ice bucket</td><td>Fisher Scientific</td><td>02-591-44</td><td>for storage of ice used during the procedure</td></tr><tr><td>liver powder</td><td>MP Biomedicals LLC.</td><td>900396</td><td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td></tr><tr><td>Microwave oven</td><td>A variety of vendors</td><td>not applicable</td><td>to prepare 2% agarose solution</td></tr><tr><td>petridish (100 x 15 mm)</td><td>Fischer Scientific</td><td>875713</td><td>interfering RNA/nanoparticle feeding chamber for larvae</td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>S220</td><td>for preparation of buffers</td></tr><tr><td>Razor blade</td><td>Fischer Scientific</td><td>12-640</td><td>to divide the interfering RNA/nanoparticle pellet for feedings</td></tr><tr><td>siRNA</td><td>Thermo Scientific/Dharmacon</td><td>custom</td><td>for preparation of siRNA/nanoparticles</td></tr><tr><td>Sodium Sulfate, Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>BDH/distributed by VWR</td><td>VWR BDH0302-500G</td><td>to prepare 50 mM sodium sulfate solution in which the interfering RNA will be resuspended</td></tr><tr><td>Thermometer</td><td>VWR</td><td>61066-046</td><td>to measure the water bath temperature</td></tr><tr><td>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><td>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 -80&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Vortex mixer</td><td>Fischer Scientific</td><td>02-216-108</td><td>for preparation of chitosan/interfering RNA</td></tr><tr><td>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

媒介蚊は、他の生物よりも多くの人的被害を与える-と百万人以上の人毎年殺す。蚊のゲノムプロジェクトは、プライマリアフリカのマラリアベクトルハマダラカとデング熱や黄熱病ベクトルネッタイシマカにおける機能遺伝子研究を含め、蚊の生物学の新しいファセットの研究を促進した。電波妨害RNA（RNAiを）媒介遺伝子サイレンシングは、これらの疾患の媒介蚊の種の両方において、目的の遺伝子を標的とするために使用されてきた。ここでは、食物と一緒にし、幼虫により摂取されたキトサン/干渉RNAナノ粒子の調製のための手順を記載している。長い二本鎖RNA（dsRNA）または低分子干渉RNA（siRNA）分子と互換性があり、この技術的に簡単で、高スループット、比較的安価な方法は、Aの異なるいくつかの遺伝子のノックダウンの成功のために使用されているハマダラカとA.ネッタイシマカ幼虫。定量RT-PCRを介して、またはin situハイブリダイゼーションで確認された幼虫授乳、ノックダウン、続いて、少なくとも後半蛹を通 ​​じて持続することができます。この方法は、農業害虫、並びに他の非モデル生物を含む蚊および他の昆虫種の多種多様に適用可能である。研究室でのその有用性に加えて、将来、キトサン、安価な、非毒性および生分解性ポリマー中に、潜在的な分野で利用することができる。

<html> A.ハマダラカ： キトサン/ dsRNAはナノ粒子によるキトサンのアミノ基とdsRNAのリン酸基との間の静電相互作用によって形成されている。溶液からのdsRNAの枯渇によって測定されるように、ナノ粒子中へのdsRNAの取り込みの効率は、通常90％以上である。原子間力顕微鏡画像は、100〜200nmの（ 図1）の範囲の直径がそのキトサンのdsRNA粒径平?...

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

ここでは、幼虫により摂取されたキトサン/干渉RNAナノ粒子の使用を通じて疾病媒介蚊で遺伝子機能を阻害するための手順を記述している。

病気のベクトル蚊幼虫におけるキトサン/干渉RNAナノ粒子媒介遺伝子サイレンシング

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 ビュー.  Kansas State University. ここでは、幼虫により摂取されたキトサン/干渉RNAナノ粒子の使用を通じて疾病媒介蚊で遺伝子機能を阻害するための手順を記述している。 

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

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

生物学

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.

篩部と半翅目昆虫の植物 sap 供給の RNA 干渉を誘発する二本鎖 RNA の経口配信方法

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

環境学

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.

再現可能な dsRNA のインジェクションと蚊とハエの産卵生物検定

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

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

蚊幼虫における遺伝子の機能解析の遺伝子配達ベクトルとして組換え蚊 Densovirus を使用

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

発生生物学

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.

ウイルス誘導ジーンサイレンシング（VIGS）でタバコbenthamianaとトマト

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

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAiによる遺伝子ノックダウンと<emインビボで&gt;</em大人の女性の&gt;利尿アッセイ<em&gt;ネッタイシマカ</em&gt;蚊

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAiのトリガ配信へ<em&gt;ハマダラカ</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

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

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA 干渉法の活用: ゴキブリのリポソーム キャリアで二本鎖 RNA の経口デリバリー

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

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.

成虫 のアノフェレスガンビアエ 蚊への効果的な経口RNA干渉(RNAi)投与

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>カイコ幼虫の遺伝子サイレンシングのためのナノ粒子媒介dsRNA摂食

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>著者スポットライト:キトサンナノ粒子の供給によるdsRNA送達によるカイコの遺伝子サイレンシング研究の進展

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

Chitosan/dsRNAナノ粒子送達によるカイコ Bombyx mori の幼虫RNA干渉

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 ...

病気のベクトル蚊幼虫におけるキトサン/干渉RNAナノ粒子媒介遺伝子サイレンシング

要約

さらに動画を探す

この動画の章

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル

篩部と半翅目昆虫の植物 sap 供給の RNA 干渉を誘発する二本鎖 RNA の経口配信方法

再現可能な dsRNA のインジェクションと蚊とハエの産卵生物検定

蚊幼虫における遺伝子の機能解析の遺伝子配達ベクトルとして組換え蚊 Densovirus を使用

ウイルス誘導ジーンサイレンシング（VIGS）でタバコbenthamianaとトマト

RNAiによる遺伝子ノックダウンと

RNAiのトリガ配信へ

RNA 干渉法の活用: ゴキブリのリポソームキャリアで二本鎖 RNA の経口デリバリー

成虫のアノフェレスガンビアエ蚊への効果的な経口RNA干渉(RNAi)投与

Chitosan/dsRNAナノ粒子送達によるカイコ Bombyx mori の幼虫RNA干渉

病気のベクトル蚊幼虫におけるキトサン/干渉RNAナノ粒子媒介遺伝子サイレンシング

要約

さらに動画を探す

この動画の章

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル

篩部と半翅目昆虫の植物 sap 供給の RNA 干渉を誘発する二本鎖 RNA の経口配信方法

再現可能な dsRNA のインジェクションと蚊とハエの産卵生物検定

蚊幼虫における遺伝子の機能解析の遺伝子配達ベクトルとして組換え蚊 Densovirus を使用

ウイルス誘導ジーンサイレンシング（VIGS）でタバコbenthamianaとトマト

RNAiによる遺伝子ノックダウンと

RNAiのトリガ配信へ

RNA 干渉法の活用: ゴキブリのリポソーム キャリアで二本鎖 RNA の経口デリバリー

成虫 のアノフェレスガンビアエ 蚊への効果的な経口RNA干渉(RNAi)投与

Chitosan/dsRNAナノ粒子送達によるカイコ Bombyx mori の幼虫RNA干渉

RNA 干渉法の活用: ゴキブリのリポソームキャリアで二本鎖 RNA の経口デリバリー

成虫のアノフェレスガンビアエ蚊への効果的な経口RNA干渉(RNAi)投与