我々は、博士に感謝しています。 SPディネッシュ-クマールとユール劉TRV - VIGSのベクトルに対して、と博士。チャックKenerley、テリーウィーラー、ジムスターと綿の種子を提供するためのバイエルクロップサイエンス社。この作品は、PHにLSとNIHにNSFによって資金を供給された

1。綿の苗を育て<ol><li>アップランド綿（チョウ目害虫抵抗性ワタ）、様々なFibermax 832、Phytogen 425RF、Phytogen 480WRおよびメトロミックス700（SunGR、Beavile、ワシントン州）を含むポット（直径7cm）でDeltapine 90の種をまく。 </li><li> 23℃プラスチックドームで覆われているトレイにポットを保つ成長室で12時間light/12時間暗光周期と° C、120μEM -2 S -1の光、。 </li><li> two子葉が浮上しているドームを取り外します。 </li><li>約2週間後、VIGSアッセイの2つの完全に展開された子葉と植物を使用してください。この段階では、真の葉はまだ（図1）浮上していない。 </li></ol> 2。 GrCLA1​​遺伝子を運ぶVIGSベクターを構築<ol><li> G.のcDNAライブラリーからプライマーGrCLA1 ​​- Fを用いるPCR、5&#39; - GCCCTTTGTGCATCTTC - 3&#39;とGrCLA1 ​​- R、5&#39; - CTCTAGGGGCATTGAAG - 3&#39;で綿のCloroplastos alterados 1遺伝子（GrCLA1）を増幅するraimondii葉組織。 </li><li> EcoRIとKpnIでGrCLA1 ​​のPCR産物を消化し ​​、ライゲーションによってpYL156（pTRV - RNA2）ベクターに挿入する。 </li><li>カナマイシンの濃度50μg/ mLを含むLB寒天プレート上でクローンをスクリーニングする。制限酵素消化およびシーケンシングにより構造を確認してください。 </li><li>エレクトロポレーションによってアグロバクテリウムツメファシエンス GV3101にプラスミドをトランスフォームし、28℃のLB液体培地で回復℃のLBとプレート+カナマイシン（50μg/ mlの）+ゲンタマイシン（25μg/ mLの）上で形質転換体を選択してください。バクテリアは、-80℃で長期使用のため25％グリセロールで保存することができます。 </li></ol> 3。 VIGS接種を行います<ol><li>カナマイシンの濃度50μg/ mLを含むLB寒天プレート上RNA2 - GrCLA1：接種の3日前、pYL192（TRV）を保持するアグロバクテリウムツメファシエンスの縞凍結グリセロールストック：RNA1、pYL156（TRV）：RNA2（ベクターのみ ​​）とpYL156（TRV）と25μgの/ゲンタマイシンのmLの。 24時間、28℃で培養する。 </li><li> VIGSの浸潤への2日前には、上記のプレートから各構成要素のために単一コロニーを採取し、カナマイシン50μg/ mLのとゲンタマイシンの25μg/ mLのを補充したLB培地5mLにそれを接種する、28で細菌培養を育て° 50rpmの速度でローラードラムで一晩のためのC。 </li><li>と20μMのアセトシリンゴン - カナマイシン50μg/ mLのとゲンタマイシンの25μg/ mLの、プラスの10mM MES（（4モルホリノ）エタンスルホン酸2）を補充したLB培地50mLでフラスコに、上記の文化を移す。 ° C〜50 rpmの速度でシェーカーで一晩のために28で文化を育てる。 </li><li>次の日に、5分間4000rpmでアグロバクテリア細胞をスピンダウンし、10mMのMgCl 2、10mMのMESおよび200μMのアセトシリンゴンを含む浸透バッファに文化を再サスペンド。 1.5文化のOD 600を調整します。 </li><li> 3時間室温でベンチに文化を残す。 </li><li> Agrobacterial浸潤に先立って、パンチ子葉を通して貫通せずに25 Gの針を持つ綿の植物の子葉の裏面。一つまたは二つの穴は、（組織の柔らかさに依存する）子葉の各セクション（図2）にパンチした。 </li><li> 1:1の比でRNA2 - GrCLA1、手には1を使用して負傷サイトを通じて子葉の下側から混合物を浸透させる：RNA1とpYL156（TRV）：：RNA2またはpYL156（TRV）pYL192のAgrobacterial培養懸濁液（TRV）を混在させるmLの無針注射器（図3）。 </li><li>プラスチックドームで植物をカバーし、一晩薄明かりの状態で、室温で浸透した植物を残す。 </li><li> 12時間の明/ 12時間暗のサイクルで23℃、120μEM -2 S -1の光の温度と成長のお部屋に植物を移す。 </li></ol>注：VIGSは、植物が23 ° Cまたは温室（25〜28 ° C）のどちらか下にあるときに動作する。温度が比較的低い場合（23〜25℃）、それは接種と表現型のためにより高い（28-30 ° C）または変動温度に比べてより均一であるほうが簡単です。ドームで植物を覆うこともVIGSがより効率的になります。 <ol start="10"><li> 7〜8日後の浸潤におけるサイレンシング表現型を調べます。 pYL156（TRV）によって沈黙植物の真の葉：RNA2 - GrCLA1 ​​はアルビノ表現型（図4）を表示するために始めた。アグロバクテリウムキャリングpYL156（TRV）を浸透植物：RNA2はコントロールとして使用。 </li><li>コントロールと沈黙綿の植物から単離されたRNAを逆転写ポリメラーゼ連鎖反応（RT - PCR）を用いて内在性遺伝子の発現レベルを調べることにより、遺伝子サイレンシングの効率を確認してください。 </li></ol>注：TRVは広い宿主範囲を持っていると届出病原体であるとしてVIGS - edの植物が含まれている検疫条件および植物の下に保持されるべきは、適切にオートクレーブし、アッセイ後に廃棄する必要があります。 4。代表的な結果： 約2週間Agrobacterial混合物と手浸潤した後、GrCLA1 ​​サイレンシングによって引き起こさアルビノ表現型が明らかに本当の葉で観察された。サイレンシングの効率は50以上の植物を使用して複数の実験でほぼ100％に達する。沈黙の植物は、新たに兄の葉（図4）上にモザイクの葉を開発する上で均一に分布し、非常に強力なアルビノの表現型を表示します。 <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="図1" /> 図 Agrobacterial浸潤のために使用される2つの完全に展開された子葉と約2週齢の段階で1。綿の苗。 <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="図2" /> 図2。Agrobacterial浸透を容易にするために、25 Gの針を使用して綿の植物の子葉の下側に小さな穴をパンチング。 <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="図3" /> 無針注射器を使用して傷のサイトを通じて、綿の子葉にAgrobacterial混合物の図3。ハンドの浸潤<img src="/files/ftp_upload/2938/2938fig4.jpg" alt="図4" /> 図4。アルビノの表現型は、綿の植物を沈黙さVIGSに登場。四品種が表示されます。 A）Fibermax 832、B）Phytogen 480WR、C）Phytogen 425RF、D）Deltapine 90。

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

利害の衝突は宣言されません。

VIGSは、一時的に内在性遺伝子の発現をノックダウンにより、機能的なゲノミクス解析の強力なツールであることが証明されています。本研究では、我々はTRV -ベースのバイナリーベクターを利用することにより、アグロバクテリウムを介したVIGSを開発した。綿CLA1（GrCLA1）遺伝子サイレンシングの効率を監視するための視覚的なマーカーとして開発されました。我々は一貫して取得し?...

<table border="1"><tbody><tr><th>試薬と機器の名称</th><th>会社</th><th>カタログ/シリアル番号</th><th>コメント</th></tr><tr><td>ローラードラム</td><td>グラス=コル、LLC。 </td><td> 099A TC108 </td><td>アグロバクテリアの培養</td></tr><tr><td>インキュベーター</td><td>シェルドンマニュファクチャリング株式会社</td><td> 01046209 </td><td>アグロバクテリアの培養</td></tr><tr><td> UV / VIS分光光度計</td><td>ベックマンコールター</td><td>モデル：DU530 </td><td>測定OD </td></tr><tr><td>ジーンパルサー</td><td> BioRad社</td><td>モデル：1652076;シリアル：154BR3880 </td><td>エレクトロポレーション</td></tr><tr><td>パルサーコントローラ</td><td> BioRad社</td><td>モデル：1652098;シリアル：232BR4833 </td><td>エレクトロポレーション</td></tr><tr><td> Micropulserエレクトロポレーションキュベット</td><td> BioRad社</td><td> 165-2081 </td><td>エレクトロポレーション</td></tr><tr><td> 1 mlのシリンジ</td><td> BDバイオサイエンス</td><td> 30962 </td><td>アグロバクテリアの接種</td></tr><tr><td>メトロミックス700 </td><td> SUNGR </td><td> SKU＃553001 </td><td>成長する苗</td></tr><tr><td>テラコッタポット</td><td> TOPlastics </td><td> GPS 3001B2 </td><td>成長する苗</td></tr><tr><td> MES一水和物</td><td> USB </td><td> 18886 </td><td>浸潤のバッファ</td></tr><tr><td>アセトシリンゴン</td><td>シグマ</td><td> D134406 </td><td>浸潤のバッファ</td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

コットン（ チョウ目害虫抵抗性ワタ ） が世界中で最も重要な作物の一つです。かなりの努力が、新品種の分子育種が行われている。綿花の大規模な遺伝子機能解析は、ゲノム、遺伝子重複と倍数性、遺伝的形質転換1〜長期成長サイクルと反抗のその大きさに起因する可能性が高い近代的な植物種、の中で最も遅れをとっている。綿の高スループット機能遺伝学/ゲノム研究を容易にするために、我々は、綿の遺伝子機能を評価するために迅速かつ効率的なトランジェントアッセイの開発を試みる。 ウイルス誘導ジーンサイレンシング（VIGS）は、ウイルス増殖2,3を抑制する宿主転写後遺伝子サイレンシング（PTGS）に基づいて開発された強力な手法です。 Agrobacterium媒介VIGSが正常に様々な機能ゲノム研究の3,4のために、広くそのようなナス科、シロイヌナズナおよびマメ科植物の種などの双子葉植物の種の範囲、及び大麦、小麦、トウモロコシを含む単子葉植物の種に適用されている。この迅速かつ効率的なアプローチは、植物形質転換を回避し、機能的な冗長性を克服するとして、それは特に魅力的と変換のための従順ではない綿のような作物種の機能性ゲノム研究に適しています。 本研究では、我々は、綿のアグロバクテリウムVIGSのシステムの詳細なプロトコールを報告する。いくつかのウイルスVIGSのベクターのうち、タバコのガラガラのウイルス（TRV）がホストの広い範囲に侵入した後、まだhosts5で軽度の症状を作り出すプラント全体にわたって精力的に感染することが可能です。サイレンシングの効率を監視するには、GrCLA1、綿のシロイヌナズナCloroplastos alterados 1遺伝子（AtCLA1）のホモログ遺伝子が、VIGSのバイナリーベクターpYL156中にクローニングし、挿入されている。CLA1遺伝子が葉緑体の開発6、および以前の研究に関与していることはことが示されているAtCLA1の機能喪失効率を黙らせるための優れた視覚的なマーカーを提供する、真の葉7のアルビノ表現型をもたらした。約2週間でポストアグロバクテリウムの浸潤、アルビノ表現型はすべて反復実験で100％のサイレンシングの効率で、真の葉の上に登場し始めました。内因性遺伝子発現のサイレンシングは、RT - PCR分析により確認した。著しく、サイレンシングが強力にテキサス州の様々な商業的に栽培品種を含めて我々がテストしたすべての品種で発生する可能性があります。この迅速かつ効率的なアグロバクテリウムVIGSアッセイは、綿のゲノムワイドなレベルでの遺伝子機能の迅速な大規模解析のための非常に強力なツールが用意されています。

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

我々は、綿のアグロバクテリウムを介したウイルス誘発遺伝子サイレンシング（VIGS）アッセイの詳細なプロトコールを提示する。タバコ茎えそウイルス（TRV）由来VIGSベクターは、R​​NAが綿GrCLA1​​、C​​loroplastos alterados 1遺伝子のサイレンシング誘導するために配備されました。サイレンGrCLA1​​によって引き起こさアルビノ表現型は、接種後2週間以内に幼苗期で観察された。

コットンでアグロバクテリウムを介したウイルス誘発遺伝子サイレンシングアッセイ

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

agrobacterium-mediated-virus-induced-gene-silencing-assay-in-cotton

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Biology

24.3K ビュー.  Texas A&M University. 我々は、綿のアグロバクテリウムを介したウイルス誘発遺伝子サイレンシング（VIGS）アッセイの詳細なプロトコールを提示する。タバコ茎えそウイルス（TRV）由来VIGSベクターは、R​​NAが綿GrCLA1​​、C​​loroplastos alterados 1遺伝子のサイレンシング誘導するために配備されました。サイレンGrCLA1​​によって引き起こさアルビノ表現型は、接種後2週間?...

ビデオ: Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

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 ...

生物学

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

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

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

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

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別 2 + ER膜のリークチャネルとその調節機序

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

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

細胞培養で誘導し、低酸素のテスト

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

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 ...

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

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

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

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

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

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

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

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

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

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

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

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

最適化と利用の<em&gt;アグロバクテリウム</em中&gt;媒介一過性タンパク質産生<em&gt;タバコ</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

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 ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

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

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

siRNA媒介サイレンシングによるマウス卵母細胞における減数分裂紡錘体の評価

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

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

コットンでアグロバクテリウムを介したウイルス誘発遺伝子サイレンシングアッセイ

要約

さらに動画を探す

この動画の章

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

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別^{2 +} ER膜のリークチャネルとその調節機序

細胞培養で誘導し、低酸素のテスト

RNAiによる遺伝子ノックダウンと

リボソーム結合した新生ポリペプチドの分離

リボソーム結合したポリペプチドを単離するためのツールとしてSECM逮捕シーケンスを使用して、

最適化と利用の

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

siRNA媒介サイレンシングによるマウス卵母細胞における減数分裂紡錘体の評価

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

コットンでアグロバクテリウムを介したウイルス誘発遺伝子サイレンシングアッセイ

要約

さらに動画を探す

この動画の章

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

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別 2 + ER膜のリークチャネルとその調節機序

細胞培養で誘導し、低酸素のテスト

RNAiによる遺伝子ノックダウンと

リボソーム結合した新生ポリペプチドの分離

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

最適化と利用の

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

siRNA媒介サイレンシングによるマウス卵母細胞における減数分裂紡錘体の評価

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別^{2 +} ER膜のリークチャネルとその調節機序

リボソーム結合したポリペプチドを単離するためのツールとしてSECM逮捕シーケンスを使用して、