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

本研究は台湾 (省の科学と技術、最も 100-2923-B-002-002-MY3、H.J.L.、106-2313-B-002-011-MY3) からの助成金によって支えられたチェコ共和国 (南ボヘミア州庁大学 GAJU 付与 065/2017/P Y.H.L) 付与、およびスペイン (スペイン経済省と競争力、CGL2012 36251 と CGL2015 64727 P コドーピング、およびカタロニアの政府への許可を付与 2014 SGR 619 コドーピングに);それはまた経済と地域開発 (コドーピング フェダーイン ・資金) の欧州基金から助成を受けた。

1 合成と dsRNA の準備
<ol><li>標的遺伝子の 3' 非翻訳領域の dsRNA のターゲット サイトを識別します。Α-チューブリン (浴槽) 遺伝子をターゲットに、dsTub を使用 (GenBank 受入番号: KX228233) と dsEGFP のシーケンスから否定的な dsRNA のコントロールは、強化された緑色蛍光タンパク質 (EGFP;GenBank の受入番号: LC311024)。</li>
	<li>T7 プロモーター配列を含む遺伝子特定のプライマーで dsRNA テンプレートを合成する標準 PCR 増幅を実行 (5'-TAATACGACTCACTATAGGG-3')。PCR 増幅条件と使用されるプライマー情報は、林らによってそれらで報告されました。(2017)12 (材料の表を参照してください)。</li>
	<li>T7 転写を用いた dsRNA 準備を進めるキット (材料の表を参照してください)、製造元の指示に従います。 注:高数量 dsRNA、(合計 40 μ L) の 1 つの管の 2 つの反応をミックスし、37 ° C で一晩インキュベートします。</li>
	<li>DNase の 2 μ L を追加 (材料の表を参照してください) に dsRNA サンプル ダイジェスト DNA テンプレートを 37 ° C で 15 分間。</li>
	<li>200 μ L の抽出試薬を追加 (材料表参照) 40 μ L のクロロホルムし渦。</li>
	<li>4 ° C で 13,000 x g で 10 分間遠心し、新しい微量遠心チューブに上清 (150 μ L) を収集します。</li>
	<li>150 μ L のイソプロパノールを追加し、氷で 15 分間インキュベート dsRNA を沈殿させます。</li>
	<li>4 ° C で 13,000 × g で 15 分間遠心し、上清を除去します。</li>
	<li>DsRNA ペレットを洗浄する 70% エタノール 200 μ L を追加します。4 ° C で 13,000 x g で 10 分間遠心</li>
	<li>エタノールを削除し、エタノールの洗浄ステップを繰り返します。</li>
	<li>エタノールと、3 分間遠心濃縮、乾燥 dsRNA ペレットを削除し、RNase フリー水で dsRNA を再懸濁します。</li>
	<li>マイクロ ボリュームが紫外可視分光光度計を使用して精製された dsRNA の数量を決定 (材料表参照) 製造元の指示に従って、必要な濃度 (例えばの準備のための 0.25 μ g/μ L に調整dsRNA リポプレックス)。</li>
	<li>3 ヵ月の-20 ° C で dsRNA のサンプルを格納します。</li>
</ol>2. dsRNA リポプレックスの準備
<ol><li>トランスフェクション試薬の 4 μ L を希釈 (材料表参照) 5% ブドウ糖溶液の 4 μ L を追加することによって。5% ブドウ糖液の 4 μ L を追加することで (1 μ g) dsRNA の 4 μ L を希釈します。</li>
	<li>希釈したリポソーム試薬溶液すぐに希釈された dsRNA のソリューションにすべて一度に追加します。簡単に、それらをミックスし、25 ° C で 15 分間インキュベートする渦 注:逆の順序でソリューションを混在させないでください。</li>
	<li>DsRNA リポプレックス使用の準備ができています。準備の 1 時間内で使用し、1 時間後に破棄します。 注:製造元の指示に従って、リポプレックスをできるだけ早く使用必要があります。</li>
</ol>3. 体液や腸ジュースから菌体外酵素のコレクション
<ol><li>
昆虫生理食塩水バッファー (10 x の在庫)
	<ol>
<li>109.3 g 塩化ナトリウム、15.7 g KCl、6.3 g CaCl2、および 8.3 g MgCl2·6H2O. 二重蒸留水 900 mL をミックスします。</li>
		<li>6.8、pH 調整、1,000 mL までを記入してください。</li>
	</ol>
</li>
	<li>
体液コレクション
	<ol>
<li>3 分の移動しないまで氷にゴキブリを麻酔します。</li>
		<li>2 本の指 (親指と人差し指) でゴキブリを押しながらゴキブリの腹側を上げます。</li>
		<li>解剖はさみを使用して、後ろ足の coxa の先端をカットします。 注:Coxa と転子間の接続交差点をカットします。Coxa の他の部分の切除は体液を収集するために効率が低下します。</li>
		<li>中指で軽く腹部を圧迫し、10 μ L マイクロ ピペットを使用して切開から出血体液を収集します。</li>
		<li>微量遠心チューブにいくつかゴキブリ (5 人) から体液をプールします。 注:マイクロ遠心チューブ用メラニン化を防ぐために氷の上を保ちます。男性のゴキブリから得られた体液量の平均は 2-3 μ L です。</li>
		<li>10、ベンチトップ遠心分離と簡単に体液をスピン s。</li>
		<li>(すなわち、10 μ L)、体液の適切なボリュームを転送し、1 x 昆虫生理食塩水バッファーの 50 μ L でそれをミックスします。</li>
		<li>血球スピンダウンに 4 ° C で 1,000 x g で 10 分間遠心します。きれいな遠心チューブに上清を転送します。</li>
		<li>A28015を測定することにより微量紫外可視分光光度計を使用して総蛋白質濃度を定量化します。</li>
		<li>同じタンパク質濃度 (6 mg/μ L の総蛋白) を持っている各サンプルを調整します。</li>
	</ol>
</li>
	<li>
腸ジュース コレクション
	<ol>
<li>氷の上のゴキブリを麻酔します。ゴキブリ腹側解剖皿の上で固定冷たい 1 x 昆虫生理食塩水バッファー昆虫ピンを使用します。</li>
		<li>高級ピンセットで腹部を解剖します。昆虫生理食塩水バッファー × 1 新鮮な料理に全体腸 (フロント、中間、および後肢腸を含む) を削除します。</li>
		<li>前部および後ろ足の腸を削除し、100 μ L 昆虫生理食塩水バッファーを含む微量遠心チューブに腸をすばやく転送します。</li>
		<li>同じ管で 10 の渦 6 ゴキブリから蚊をプール s。</li>
		<li>スピン ・ ダウン血液細胞と腸の組織に 4 ° C で 1,000 x g で 10 分間遠心します。きれいな遠心チューブに上清を転送します。</li>
		<li>A28015を測定することにより微量紫外可視分光光度計を使用して総蛋白質濃度を定量化します。</li>
		<li>同じタンパク質濃度 (6 mg/μ L の総蛋白) を持っている各サンプルを調整します。</li>
	</ol>
</li>
</ol>4. dsRNA 劣化分析
<ol><li>新鮮な裸の dsRNA の dsRNA リポプレックス (1 μ g) 4 μ L を準備します。</li>
	<li>体液、または腸から dsRNA 抽出ソリューション昆虫生理食塩水バッファー (コントロール) の 10 μ L の酵素ジュースをミックスします。</li>
	<li>2 μ L の酵素活性を阻害するコントロールとして試料を分離するグリコールエーテルジアミン四酢酸 (20 mM) または RNase フリー水のいずれかを追加します。</li>
	<li>1 時間 (6、12、または 24 時間以上) 25 ° C でインキュベートします。</li>
	<li>200 μ L の抽出試薬と 40 μ L のクロロホルム、渦、追加します。</li>
	<li>4 ° C で 13,000 x g で 10 分間遠心し、新しい微量遠心チューブに上清 (150 μ L) を収集します。</li>
	<li>150 μ L のイソプロパノールを追加し、氷で 15 分間インキュベート dsRNA を沈殿させます。</li>
	<li>4 ° C で 13,000 × g で 15 分間遠心し、上清を除去します。</li>
	<li>DsRNA ペレットを洗浄する 70% エタノール 200 μ L を追加します。4 ° C で 13,000 x g で 10 分間遠心</li>
	<li>エタノールを削除し、エタノールの洗浄ステップを繰り返します。</li>
	<li>エタノール、10 μ L の RNase フリーの水で 3 分再懸濁します dsRNA の遠心濃縮、乾燥 dsRNA ペレットを削除します。</li>
	<li>1.5% の agarose のゲルの電気泳動によって扱われた dsRNA の整合性をチェックします。</li>
</ol>5. dsRNA の内服
<ol><li>ドライフード (犬の食事) の自由食事にゴキブリを維持します。DsRNA 摂取実験の前日、ゴキブリから水の供給を奪います。</li>
	<li>フレキシブル鉗子 (図 1、ステップ 5) でその翼を掴んでゴキブリを保持します。 注:ゴキブリの体の部分をつかむか。</li>
	<li>セクション 2 の説明に従って、フィード dsRNA リポプレックスとして裸の dsRNA を準備します。 注:前に餌、dsRNA リポプレックスをたて準備されるべき。</li>
	<li>フィードの dsRNA リポプレックスまたは裸の dsRNA の 4 μ L (250 ng) マイクロ ピペットで。</li>
	<li>ゆっくりピペット dsRNA ソリューションの液滴、ゴキブリの口器に近いし、ゴキブリは完全に液滴を摂取します。</li>
	<li>8 日か 16 日間連続 (1 h 点灯後、消灯前に 1 h) は 1 日 2 回の摂取実験を実行します。 注:ゴキブリは水だけ摂取実験中に dsRNA のソリューションまたはコントロール試薬 (例えば、ヌクレアーゼ フリー水、ブドウ糖溶液、リポソーム試薬) とマニュアル給餌を取得しました。</li>
	<li>ゴキブリに dsRNA 実験の摂取の 16 日後の水のボトルを提供します。</li>
</ol>6. ノックダウンを評価します。
<ol><li>選択した時にポイント (dsRNA 摂取試験後 2、9、17 日) は dsEGFP と dsTub 扱われる昆虫を収集そして選ばれた組織 (例えば腸) を解剖します。</li>
	<li>量的なリアルタイム PCR (qRT PCR) を実行します。QRT PCR 増幅条件およびプライマー情報林らによって報告されました。(2017)12。</li>
	<li>コントロールと死亡を確認し、移動は、もはやゴキブリを削除は毎日昆虫 dsRNA 治療を評価します。</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+lipid+nanoparticles+for+siRNA+delivery.">Advances in lipid nanoparticles for siRNA delivery.</a> Pharmaceutics. 5, 498-507 (2013).</li><li>Xia, Y., Tian, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+surface+properties+on+liposomal+siRNA+delivery.">Effect of surface properties on liposomal siRNA delivery.</a> Biomaterials. 79, 56-68 (2016).</li></ol>

著者が明らかに何もありません。

このプロトコルは、ドイツのゴキブリの中腸ジュースのリボヌクレアーゼ消化に対する保護を含む dsRNA リポプレックスの口頭配達を通じて効果的な RNAi のメソッドを示します。様々 な昆虫の種の他の研究のように、dsRNA8,9,10の劣化による dsRNA の口頭配達を通じて貧しい RNAi 効果がほとんどを占めています。このプロトコ?...

RNAi は、多くの真核生物1の dsRNA の分子によってトリガーされる転写後サイレンシング経路の機構を通じてノックダウン遺伝子発現する効果的な方法として実証されています。過去の研究では、RNAi オゾン注入を介して特定の遺伝子発現および/または昆虫2,3のさまざまな分類群の dsRNA の供給によって動作する開発から遺伝子の機能を研究するための有用なツールとなっています。特異性と破壊効果の頑健性のため、RNAi のアプリケーションは現在害虫制御管理4、5のための潜在的な戦略として検討されています。ただし、RNAi の効率は昆虫種間対象別の遺伝子と配信方法によって異なります。証拠の成長するボディは、リボヌクレアーゼによる劣化は、dsRNA の不安定性 RNAi5,6の限られた有効性の重要な要因であることを示唆しています。例えば、 Manduca sextaの RNAi の感度が低いは、体液混合 dsRNA が 1 時間7内ですぐに劣化したという事実によって説明されています。同様に、効率的に摂取された dsRNA が低下する、中腸でアルカリ性の核酸分解酵素の存在の異なる昆虫8,9,10で RNAi 効率が低い相関が強い。
DsRNA の口頭配達は害虫制御戦略の RNAi のアプリケーションの特に興味深いですが中腸における核酸による dsRNA の劣化を遅らせる方法はまだ開発されていない、有効なことを確認する可能性のあるだろうRNAi を介して供給します。ただし、dsRNA、カイコ、例えば50 μ g の大量の餌やイナゴ種で 8 日間 (合計 8 dsRNA μ g)、継続的に供給で dsRNA の口頭配達する RNAi の応答が報告されています。チャバネゴキブリ germinicaチャバネゴキブリが dsRNA11,12,13,14、注入による RNAi に敏感を介して供給 dsRNA に敏感ではないです。最近では、林ら(2017) は dsRNA カプセルこと成功した rnai ノックダウンするリポソーム キャリア結果とα-チューブリン遺伝子発現15チャバネゴキブリの中腸とトリガー有意な死亡率を示しています。中腸における dsRNA の劣化は、口腔の RNAi のための制限要因は、リポソーム キャリア劣化は、腸内で強いヌクレアーゼ活動とその他の昆虫に適用が容易ですから dsRNA を保護する手段として役割を果たします。注特定のトランスフェクション試薬を選ぶ理由の現在のプロトコルのリポソーム キャリアはそれは毒性が少なく、昆虫の細胞ラインの transfection のためテストされていますを使いました (材料の表を参照) によると、節してください。Gharaviらの異なるリポソーム トランスフェクション システムとの比較によって(2013)16、株小さい干渉の RNA (siRNA) の効率はおよそこれと他の昆虫の17,18 dsRNA 配信システムに使用されている他の市販システム間で同じ.さらに、私たちの供給法は各ゴキブリで dsRNA の適切な量を摂取し、結果が得られる堅牢で確認をように十分注意してください。要約すると、議定書と結果を示す、dsRNA リポプレックスを使用して dsRNA の安定性を向上させる将来的に害虫駆除のための有望なアプローチである、RNAi の戦略の口頭配達のデザインへの扉を開きます。

<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 干渉 (RNAi) 多数の遺伝子の生物学的機能を暴くため広く用いられてきた、重要な遺伝子発現の中断動作害虫コントロール ツールとして想定されました。二本鎖 RNA (dsRNA) を正常に配信のインジェクション、給餌と、浸漬など、さまざまな方法が報告されている dsRNA の経口デリバリーによる RNAi の効率は非常に異なる昆虫グループ間で可変。チャバネゴキブリチャバネゴキブリは、以前発表された多くの研究で示されている dsRNA の注入に非常に敏感。本研究では、リポソーム キャリアとカプセル化された dsRNA が腸ジュースで dsRNA の劣化を遅らせるに十分であることを示す方法について説明します。とりわけ、大幅リポソームによってカプセル化された dsRNA の連続的供給、中腸におけるチューブリンの発現を減少させる、ゴキブリの死をもたらした。結論としては、定式化と核酸に対して dsRNA を保護する dsRNA リポプレックスの利用かもしれない害虫制御のための RNAi の実用的な使用、将来的に。

DsRNA の経口配信用プロトコルの簡易方式はで示した図 1、dsRNA リポプレックスの準備のため、主要な手順が表示されます。
チャバネゴキブリ、dsTub リポプレックス腸ジュースで培養を行った前のヴィヴォアッセイおよび dsRNA の整合性の中腸ジュースの dsRNA の劣化にリポソーム キャリア...

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

この原稿は、リポソームにカプセル化された二本鎖 RNA の経口摂取によってドイツのゴキブリの中腸における遺伝子発現の減少を示しています。

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 ビュー.  National Taiwan University. この原稿は、リポソームにカプセル化された二本鎖 RNA の経口摂取によってドイツのゴキブリの中腸における遺伝子発現の減少を示しています。

ビデオ: 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.

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、<em&gt; Dermestes maculatus</em

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

遺伝学

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 ...

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.

特定の発生時における遺伝子発現を操作するための強力なツールとしての水生カブトムシのRNA干渉

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.

アメリカのゴキブリにおけるRNA干渉の応用

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

発生生物学

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.

dsRNAをインジェクションによるRNAiの干渉にショウジョウバエ胚

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.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

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.

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 ...

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 ...

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

要約

さらに動画を探す

この動画の章

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、

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

特定の発生時における遺伝子発現を操作するための強力なツールとしての水生カブトムシのRNA干渉

アメリカのゴキブリにおけるRNA干渉の応用

dsRNAをインジェクションによるRNAiの干渉にショウジョウバエ胚

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

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

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

要約

さらに動画を探す

この動画の章

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、

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

特定の発生時における遺伝子発現を操作するための強力なツールとしての水生カブトムシのRNA干渉

アメリカのゴキブリにおけるRNA干渉の応用

dsRNAをインジェクションによるRNAiの干渉にショウジョウバエ胚

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

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

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