作業は、助成金なしでサポートされていました。 &quot;BundesministeriumこだわりForschungウントTechnologie（BMBF）の01GU0615。

shRNAの1）細菌配達<ol><li>細菌培養を開始する前に、一つはLB -培地およびLB -寒天を準備する必要があります。 </li><li> LB -培地は、酵母エキス（0.5％w / v）の、バクトトリプトン（1.0％w / v）を、およびNaCl（0.6％w / v）の検量するとアクアbidestでこれらのコンポーネントを希釈するため。調製された溶液は、オートクレーブで滅菌する必要がありますし、使用する準備が整いました。 </li><li> LB -寒天プレート用バクト寒天（1.5％w / v）を滅菌する前のLB -培地に添加する必要があります。 </li><li> LB -寒天が完全に溶解するまで電子レンジでLB -寒天を加熱する。ボトルが火傷することなく簡単に触れることができるまで溶液が冷却させます。さらなるステップで陽性クローンの選択のためのカナマイシン（100 mg / mL）を追加します。 </li><li> 10cmのペトリ皿にLB -寒天溶液20mlをピペッティングすることにより、層流ベンチ（無菌状態）でLB -寒天プレートを準備します。液体が固体になるまでのプレートが冷却させます。プレートは使用可能になりますし、4℃で保存することができます。 </li><li> shRNAをコードする発現ベクターはコンピテント大腸菌に変換されますCaCl 2の手順を使用して、熱ショックにより大腸菌 ceq221。 </li><li>板100μg/ mlのカナマイシンを含むLB -寒天プレート上で形質転換された細菌は、、蓋を閉じてパラフィルムでプレートをシールし、℃で一晩37℃で逆さまに栽培しています。 </li><li>歯のピックで生育したコロニーを選択することにより陽性クローンを持つ細菌のミニ培養を接種する。歯が100 mg / mlのカナマイシンおよび° C一晩200 rpmで振とう機で37℃で栽培を含む新鮮なLB -培地の7.0 mlにピックアップ移す。 </li><li> 1リットルの三角フラスコに一晩ミニカルチャー、100 mg / mlのカナマイシン1:100を含むLB -培地新鮮な100mlを植菌し、200rpmで° C一晩シェーカー上で37インキュベートする。 </li><li>シード25 × 10 4、6のウェル当たりヒト胃癌細胞（EPG85 - 257RDB）（播種した細胞の数に従って、細胞株の細胞増殖の速度に依存する細胞集密度は24時間播種後〜70から80パーセントにする必要があります） -よく料理し、37これらをインキュベート℃、一晩5％CO 2、水蒸気飽和雰囲気インチ</li><li> TKのRNAiベクターを含有E.の夜の文化上、癌細胞の感染の前大腸菌は、OD 600を決定するために光度計で測定されています。 0.5の光学密度まで、菌液を希釈する（OD 600 = 0.5は1.6 × 10 8細菌細胞/ mlに等しい）に達している。 </li><li> FCSを含まない培地の共同インキュベーション細菌へ前の30分に対して、癌細胞のFCSを含む細胞培養培地を交換してください。 </li><li> 1 × PBSで2回細菌を洗浄し、無血清リーボビッツL - 15培地で希釈する。 </li><li> 37℃で2時間ガン希望のMOIで細胞（癌細胞あたりの細菌細胞の感染多重度は=数）と共同インキュベーション細菌やがん細胞に希釈した細菌を追加℃を</li><li>共同インキュベーションの癌細胞の2時間は、100 U / mlのペニシリン、100μg/ mlのストレプトマイシン、2.5μg/ mlのアンホテリシン、150μgの/を添加した血清含有リーボビッツL - 15培地で1回1 × X PBSで2回洗浄し、された後にミリリットルゲンタマイシン、および100μg/ mlのカナマイシン。 </li><li> 37℃で癌細胞を培養° Cを、5％CO 2、水蒸気飽和雰囲気の中で継続する。 </li><li>細菌治療の効果は、mRNAレベルで定量的リアルタイムRT PCRによって測定された蛍​​光顕微鏡、、でおよびタンパク質レベルでのウェスタンブロット解析によって視覚化、およびFACS分析、および細胞増殖アッセイのような機能的アッセイで表示できます。 （したい場合は、これらの手順を詳細に説明することができます）。 </li></ol> 2）代表者の結果プロトコルが正しく行われている場合、結果は以下に示すものと同等でなければなりません。 蛍光顕微鏡図1は、三時間細菌の治療後のヒト胃癌細胞を（）未処理細胞（B）と比較して示しています。処理した細胞の核の周りに、細菌を検出することができます。 <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="図1" /> 図1：細菌の治療（1:500）の後に蛍光顕微鏡でヒト胃癌細胞と未処理ヒト胃癌細胞制御として、DAPI染色、DAPIバンドパスフィルタ（Λemは= 640 nm）を、40倍目標。 定量的リアルタイムRT PCR 図2では約70％（黒線）のMDR1 mRNAのダウンレギュレーションは、治療上の細菌による治療後に見ることができます。親細胞はMDR1の過剰発現の欠如に起因するポジティブコントロールとして使用。細胞株を発現するプラスミド抗MDR1したshRNAの他のRへtranskingdomのRNAi技術の直接比較として撮影を含む257RDB p170NAIは、戦略をサイレンシング。未治療耐性細胞株EPG85 - 257RDB過剰発現するMDR1、発現するプラスミドshRNAを欠いている細菌で処理同じ細胞株、およびこの細胞株を発現するプラスミド抗MRP2 shRNAを運ぶの治療細菌による治療は、陽性対照として採用した。 <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="図2" /> 図2：治療E.治療後の定量的リアルタイムRT PCR MDR1 mRNA発現抗MDR1 shRNAを（：500 MOI 1）を発現する大腸菌 ceq221。正規化は、ハウスキーピング遺伝子のアルドラーゼを用いて行った。細胞株EPG85 - 257RDBの未処理の細胞のMDR1/aldolase比率が100％に設定されていました。 P -値はスチューデントのt検定を使用して計算された（* = p &lt;0.05、** = P &lt;0.005、*** = p &lt;0.001）。 ウェスタンブロット分析 図3は、ダウンレギュレーションMDR1も細菌の治療後のタンパク質レベルで行われたことがわかります。それは、薬剤に敏感な親細胞系（EPG85 - 257P）、未治療の薬剤耐性細胞株（EPG85 - 257RDB）のMDR1の発現と処理されたサンプルのMDR1発現の欠けているMDR1の発現を示しています。バクテリア処理した細胞のMDR1の明確なダウンレギュレーションが観察することができます。 <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="図3" /> 図3：ウエスタンブロット分析の未治療の薬剤感受性EPG85 - 257PのMDR1発現レベル、E.との共同インキュベーション後の未治療の薬剤耐性EPG - 257RDB、およびEPG85 - 257RDBの。 大腸菌 ceq221 + P43 MDR1。一次抗体C219 1:100、及び抗アクチン1時05分000、二次抗体抗マウス1:10000。 細胞毒性分析図4に示すように細胞毒性アッセイのような機能解析もtranskingdom RNAiの機能のための指標です。親細胞株とを発現するプラスミドANIT - MDR1 shRNAを含む細胞株は、ダウノルビシンへの抵抗性を示していない。耐性細胞株EPG85 - 257RDBと、さらに2つのコントロールは、ダウノルビシンに対する抵抗が約90％で逆転することができる抗MDR1 shRNAを発現している細菌で処理された試料に比べて抵抗値の有意な変化を示していない。 <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="図4" /> 図4：細胞毒性アッセイ。薬剤特異的細胞の生存のための細胞毒性アッセイによって決定されたIC50 -値。 P -値はスチューデントのt検定を使用して計算された（* = p &lt;0.05、** = P &lt;0.005、*** = p &lt;0.001）。 アントラサイクリン系薬剤の蓄積アッセイ図5に示すように、バクテリア処理した試料の抵抗力低下（図4）によると、抗MDR1 shRNAを処理した細胞のアントラサイクリン蓄積が約90％増加しています。親細胞系と細胞株257RDB p170は100％に強いダウノルビシン蓄積を示す。耐性変異株はほとんどあらゆる蓄積を示していない。抗MDR1 shRNAを発現している細菌で処理した細胞は90％のanthracyline蓄積の増加を示す。 <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="図5" /> 図5：アントラサイクリン蓄積アッセイ。 6日間をフローサイトメトリーで測定した細菌の治療後の癌細胞のアントラサイクリン蓄積。 P -値はスチューデントのt検定を使用して計算された（* = p &lt;0.05、** = P &lt;0.005、*** = p &lt;0.001）。

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

利害の衝突は宣言されません。

使用される感染症とそれに対応するMOIための播種細胞数は、批判的に細胞観察の下に線との成長の速度に依存する。シードに最適な細胞数を検索するには、事前に実験の成長の速度を決定することが強く推奨されています。このほかに、別の湿気が原因で細胞がストレスで死ぬことなく、細菌の侵入に耐えることができるためにどの拡張限定にテストする必要があります。観察下にある遺伝?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
<td>&nbsp;</td>		</tbody>
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bacterial delivery of rnai effectors: transkingdom rnai

RNA干渉（RNAi）は、mRNA発現の特異的阻害のための高い効果的なメカニズムを表します。強力な実験ツールとしての可能性に加えて、RNAi経路は、治療の利用のために有望なように見えます。 RNA干渉（RNAi）ベースの治療法の開発のため、標的細胞へのRNAiを媒介剤の送達は、大きな障害の一つです。このハードルを克服する新たな戦略は、transkingdomのRNAi（RNA干渉TK）です。この技術は、生産し、RNAiを誘導する標的細胞への治療短鎖ヘアピンRNA（shRNA）を提供するために、非病原性の細菌、例えば大腸菌（Escherichia coli）を使用しています。第一世代のTK RNAiを媒介ベクトル、TRIPは、関心の治療shRNAの発現調節のためのバクテリオファージT7プロモーターが含まれています。また、TRIPは、この酵素listeriolysin Oを生成するリステリア菌からβ1-インテグリン陽性哺乳類細胞とHlyAの遺伝子を、入力するように自然な非侵襲的な細菌を許可するインベイシンをコードエルシニア偽結核からInv遺伝子座を、持って治療したshRNAがから脱出することができますエントリは、標的細胞の細胞質内小胞。 TRIPコンストラクトはshRNAのT7プロモータードリブン合成に必要なT7 RNAポリメラーゼをコードして有能な非病原性大腸菌の菌株に導入されています。異なるRNAiの戦略のための十分に特徴付けられた癌関連標的分子は、ABCB1（MDR1/P-glycoprotein、MDR1/P-gp）です。薬剤の押し出しポンプとを仲介する&quot;古典的&quot;ABCB1を介した多剤耐性（MDR）特定の交差抵抗性パターンによって特徴付けられるヒト癌細胞の表現型としてこのABCトランスポーターの役割を果たします。異なるABCB1発現MDR癌細胞を抗ABCB1 shRNAの発現ベクターベアリングE.で処理した大腸菌 。この手順では、がん細胞とABCB1コードmRNAと同様に対応する薬剤の押し出しポンプのかなりのダウンレギュレーション内のRNAi経路の活性化をもたらした。したがって、薬物の蓄積は、原始的な薬剤耐性癌細胞で強化され、MDRの表現型を逆転した。このモデルにより、データはtkの RNAiがヒト癌細胞における癌関連因子、例えば、ABCB1、の変調に適していることが証明の概念を提供しています。

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA干渉（RNAi）ベースの治療法の開発のための、新たな戦略が開発された、transkingdomのRNAi（tkRNAi）。この技術は、生産し、標的細胞への治療短鎖ヘアピンRNA（shRNA）を提供する非病原性細菌を使用しています。ここで、tkRNAiが正常に癌細胞の古典的なABCB1を介した多剤耐性（MDR）の反転を適用した。

RNAiのエフェクターの細菌配達：Transkingdom RNAiの

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

bacterial-delivery-of-rnai-effectors-transkingdom-rnai

Research

JoVE Journal

Biology

13.4K ビュー.  Charité Campus Mitte. RNA干渉（RNAi）ベースの治療法の開発のための、新たな戦略が開発された、transkingdomのRNAi（tkRNAi）。この技術は、生産し、標的細胞への治療短鎖ヘアピンRNA（shRNA）を提供する非病原性細菌を使用しています。ここで、tkRNAiが正常に癌細胞の古典的なABCB1を介した多剤耐性（MDR）の反転を適用した。

ビデオ: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

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

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

ローカライズされたRNAiと創薬リーチにおける外来遺伝子の発現が

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

酵母の菌タイプIIIエフェクターの発現により誘導される増殖阻害表現型の同定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

哺乳動物細胞におけるRNAiスクリーニングのためのミッションesiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAiは、Necromenic虫にマイクロインジェクションによる遺伝子ノックダウンとトランスジェネシスを媒介 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

の定量と自動ハイスループットゲノムワイドRNAiスクリーニング C.エレガンス</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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 ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

齧歯類精巣上体精子のリン酸化ペプチドの分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...