我々は彼らの貢献のためにD. Braendle、CLクルツとF.Montañana·サンチスに感謝します。このプロジェクトは、ANRとConseilの地域PACAと、INSERMおよびCNRSからの制度資金からの補助金によってサポートされていました。

<ol>
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K., Miteva, Y., Hannenhalli, S., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physiological+activation+of+osmosensitive+gene+expression+mimics+transcriptional+signatures+of+pathogen+infection+in+C.+elegans.">Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans.</a> PLoS One. 5, e9010-e9010 (2010).</li><li>Lee, K. Z., Kniazeva, M., Han, M., Pujol, N., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fatty+acid+synthase+fasn-1+acts+upstream+of+WNK+and+Ste20&#47;GCK-VI+kinases+to+modulate+antimicrobial+peptide+expression+in+C.+elegans+epidermis.">The fatty acid synthase fasn-1 acts upstream of WNK and Ste20&#47;GCK-VI kinases to modulate antimicrobial peptide expression in C. elegans epidermis.</a> Virulence. 1, 113-122 (2010).</li><li>Duverger, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automated+high-throughput+approach+to+the+generation+of+transposon+insertion+mutants+in+the+nematode+Caenorhabditis+elegans.">A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.</a> Nucleic Acids Res. 35, e11-e11 (2007).</li><li>Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20982-20987 (2008).</li><li>Ruzanov, P., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+SAGE+analysis+of+the+Caenorhabditis+elegans+transcriptome.">Deep SAGE analysis of the Caenorhabditis elegans transcriptome.</a> Nucleic Acids Res. 38, 3252-3262 (2010).</li><li>Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+in+a+dish:+high-throughput+analyses+of+bacterial+pathogenesis.">Infection in a dish: high-throughput analyses of bacterial pathogenesis.</a> Curr. Opin. Microbiol. 10, 10-16 (2007).</li><li>Okoli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+antifungal+compounds+active+against+Candida+albicans+using+an+improved+high-throughput+Caenorhabditis+elegans+assay.">Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay.</a> PLoS One. 4, e7025-e7025 (2009).</li><li>Moy, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 10414-10419 (2006).</li><li>Ballestriero, F., Thomas, T., Burke, C., Egan, S., Kjelleberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+compounds+with+bioactivity+against+the+nematode+Caenorhabditis+elegans+by+a+screen+based+on+the+functional+genomics+of+the+marine+bacterium+Pseudoalteromonas+tunicata+D2.">Identification of compounds with bioactivity against the nematode Caenorhabditis elegans by a screen based on the functional genomics of the marine bacterium Pseudoalteromonas tunicata D2.</a> Appl. Environ. Microbiol. 76, 5710-5717 (2010).</li></ol>

ユーバンクラボでは、連合BiometricaとMODULバイオと協力しています。

<p class="jove_content"&gt;現在のプロトコルは、大規模なRNAiスクリーニングでできます。<em&gt; C.エレガンス</em&gt;読み出しとして蛍光レポーター遺伝子の発現レベルを使用する固体培地上で。それは、効率的で信頼性と再現可能な方法で、多数のサンプルを簡単かつ高速処理を可能にする、ハイスループットアッセイを容易にする自動化ツールで最適化されています。他の方法は、自動液体試料の取り扱い上のように大きく依存していないことが記載されている<sup&gt; 8</sup&gt;。</p&gt;<p class="jove_content"&gt;プロトコルの1つの重要な組織的な側面は、企業のModulBioと共同でRNAiスクリーニング手順に商用MBioLIMSソフトウェアを適応させることによって、サンプルトラッキングの堅牢な方法の開発がされています。手続きのあらゆる段階で、プレートは今LIMS内で生成されたバーコードによって識別されます。 LIMSまた、各プレートに各ウェルのクローンのIDを記録するように、ウェルから推定される遺伝子に行くのは簡単です。そして、これを越えて、コミュニティデータベースWormBaseへLIMSのリンクは、任意の遺伝子についての分子的および機能的なデータへの直接アクセスを可能にします。 LIMSはまた、プレート、あるいは染色体と全ゲノムレベルで実施する分析を容易にします。これらの分析は、データはそのようなCOPAmulti前述したようにツールと完全に互換性のある方法で格納されている<sup&gt; 16</sup&gt;。</p&gt;<p class="jove_content"&gt;メソッドの1つの重要な実験点は、ワームの数に比例し、食品の量の調整です。多くのアッセイのために、それはワームは実験の最後に餓死しないことが不可欠であるが、それは開始時にあまりにも多くの食べ物を持っていないようにすることも重要です。我々は、食品の高密度は、pを運ぶ虫を用いたプロトコルで蛍光誘導を減少させることを観察した<em&gt; NLP-29</em&gt; :: GFPレポーター。これは、ワームはあまりにも多くの細菌（BS、未発表の結果）で培養され減少した感染効率を反映するかもしれません。これは、dsRNAは最適なレベルで生産されているかどうかを決定するので、同時に、RNAiの細菌の培養条件は、も重要です。大規模なRNAiのためのいくつかのプロトコルは、液体培養物を播種する前に、寒天プレート上でのRNAiクローンの培養を必要とする<sup&gt; 8</sup&gt;。我々はこのステップを省略して堅牢な遺伝子サイレンシングを得た。我々は、同種の結果を確実にするためにそれぞれの文化の過剰成長を選んだ。事実で説明されたプロトコルは、並列に2つまたは3つのスクリーンを行うのに十分な材料を提供する。最も堅牢な結果を得るの利益のために、我々は、ワーム、RNAiの細菌や真菌胞子の独立した培養物を使用して複製の画面を行って、しかし、支持している。</p&gt;<p class="jove_content"&gt;もう一つの重要なステップは、あまりにも高温多湿でも乾燥しすぎでもない媒体を使用してに関連しています。プレートは十分に乾燥されていない場合は、RNAiの細菌が適切に乾燥させないため、ワームは、アッセイ中に泳ぐことになります。これは問題を引き起こす可能性があります。たとえば、ワームは効率的に感染されていません<em&gt; D. coniospora</em液体中に&gt;（C. Couillault、未発表結果）。一方、乾燥したプレートに、培地の浸透圧が増加する。これは、ワームの生理に直接影響を持っている<sup&gt; 17</sup&gt;など、いくつかの抗菌ペプチド遺伝子の誘導など、<em&gt; NLP 29</em&gt;<sup&gt; 18</sup&gt;。ワームや胞子を分配するために使用されるボリュームは、効果的かつ迅速な乾燥を可能にすることが重要です。液体以上の10μLを新鮮プレートに添加されている場合は、井戸が乾燥するために多くの時間を取ることができます。これは、プレートにワームを追加するには、ロボットのメソッドを使用することは困難であることを意味します。たとえば、各ワームは、液体の約1μLでBiosortによって分配される。それはまた、私たちは堆積物の溶液中でのワームの傾向を克服することができますように我々は現在、手動でワームを配布しています。</p&gt;<p class="jove_content"&gt;我々は以前に1つの96ウェルプレートを36分で分析することが許可されてBiosortの調整を報告<sup&gt; 19</sup&gt;。マシンのメーカーUBIは現在さらに分析時間を短縮するBiosortの修正を提供しています。 &quot;FastReFlex&quot;で、試料を直接、バブルトラップフィルタをバイパスして、分析用フローセルを介してルーティングされます。データ収集のための時間の大幅な削減この結果、全体のプレートは、1日あたり処理すべき20枚の周りにできるように22分で解析することができます。プレートは凍結されていない場合は、しかし、最初と最後のものの間で8時間の間隔があるでしょう。わずか18時間持続感染実験の比較的短い期間、与えられた、これは板を越えて受け入れられない変数を導入する。分析前に凍結プレートは、この問題を解決するが、2人は簡単に50プレート週まで処理することができ、現在のプロトコルおよび標準液体ハンドリングロボットとして、それはまた、ハイスループット分析のコンテキストで不可欠であるだけでなく。現在、我々は定期的に全ゲノムの画面を可能にする30枚の週を、画面未満の2ヶ月に一度実施することができます。</p&gt;<p class="jove_content"このプロトコルの&gt;一つの興味深い機能は、液体培養では実行できませんアッセイを可能にする、固形寒天を使用している事実である。ワームの生理学では、培養条件に応じて異なりますので<sup&gt; 20,21</sup&gt;、固体培地上でアッセイを行うことができることは、またいつもの液体ベースの画面を補完することができます。特定の表現型（例えば運動）の場合、解析は96ウェル固体培地プレートで直接行うことができます。現在の画面は、しかし、Biosortのような固体培地プレートからのサンプルができない、ワームが最初に凍結する前に、液体に転送されます。一方、COPAS Biosortは多くの異なった表現型の測定との互換性multiparametric定量分析を可能にします。それは、任意の画面にすべての測定パラメータは、プライマリ読み出しされる可能性がある。それにもかかわらず、これらの測定は、いくつかの理由で役立ちます。彼らは1つがアッセイが正しく実行されたかどうかを判断することができます。例えば、一つは、各ウェル中のワームの数とサイズを確認することができます。 RNAiのクローンは、成長や繁殖を阻止すべき唯一の場所が期待値からの偏差である必要があります。 20未満のワームおよび/または30％小型動物（TOF）とウェルズは、標準に比べて成長に影響はありません標準コントロール（<em&gt;例えば、GFP（RNAi実験</em&gt;））は、以降の分析から除外することができます。それらの開発がうまく進んでいる後にこれらのクローンの機能は、ワームがRNAiの細菌にさらされている請求項代替プロトコルを使用して対処することができます<sup&gt; 15</sup&gt;。別のパラメータはまた、唯一の緑色蛍光が赤色ではなく影響を及ぼしているものにヒットを制限するたとえば、候補遺伝子（ヒット）選択を絞り込むために使用することができます。ヒットを選択するために選ばれた正確な方法は、画面の種類とその正確な読み出しに依存します。定量的なゲノムワイドな画面から生成されたデータの複雑さを考えると、統計学者とのコラボレーションが必要な場合があります。</p&gt;<p class="jove_content"&gt;我々が記述するプロトコルは、容易にアッセイの他のタイプに適合させることができます。たとえば、感染段階で<em&gt; D. coniospora</em&gt;固体培地に感染することができ、他の病原体によって置き換えることができます。我々は液体培養でセラチア感染の重要な病原因子の多くは、固形培地（E. Pradel、私信）に感染時に役割を果たしていないことを発見したので、これは潜在的に有用である。プロトコルはまた、化学化合物のテストと完全に互換性があります<sup&gt; 22から24</sup&gt;、または細菌の異なるライブラリを使用して生物活性を有する化合物を識別するために<sup&gt; 25</sup&gt;そうでは一般的なユーティリティを見つける必要があります<em&gt; C.エレガンス</em&gt;研究コミュニティ。</p

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> BactoAgar </td><td> BD診断システム</td><td> 214010 </td><td></td></tr><tr><td>アンピシリン</td><td> Sigma-Aldrich社</td><td> A9518 </td><td></td></tr><tr><td>バクトペプトン</td><td> BD診断システム</td><td> 211677 </td><td></td></tr><tr><td> CaCl 2を </td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> AeraSeal接着フィルム</td><td> Dutscher </td><td> 760214 </td><td></td></tr><tr><td>イソプロピルβ-D-チオガラクトピラノシド（IPTG） </td><td>サーモフィッシャーサイエンティフィック</td><td> AB-0481 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> MgSO 4で </td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> NaClを</td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td>のNa 2 HPO 4 </td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> NaOHで</td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> 96ディープウェルプレート</td><td>サーモフィッシャーサイエンティフィック</td><td> AB-0932 </td><td></td></tr><tr><td> 96ウェル平板</td><td> FALCON </td><td> 353072 </td><td></td></tr><tr><td> 96ウェル丸プレート</td><td> FALCON </td><td> 353077 </td><td></td></tr><tr><td>テトラサイクリン</td><td> Sigma-Aldrich社</td><td> T8032 </td><td></td></tr><tr><td>トリトンX-100 </td><td>すべてのサプライヤー</td><td></td><td></td></tr><tr><td> TECANロボット</td><td> TECAN </td><td></td><td></td></tr><tr><td> Liquidator96 TM </td><td>レイニン</td><td></td><td></td></tr><tr><td> COPAS Biosort </td><td>組合Biometrica </td><td></td><td></td></tr><tr><td> LIMS </td><td> MODULバイオ</td><td></td><td></td></tr></tbody></table>

quantitative and automated high-throughput genome-wide rnai screens in c. elegans

RNA干渉は、全ゲノム規模でかつ定量的文脈で実施する場合は特に、遺伝子の機能を理解するための強力な方法です。 C.で虫 、遺伝子の機能は、特定の遺伝子の1に対応するdsRNAを発現する細菌で、ワームを供給することにより、簡単かつ効率的にノックダウンすることができます。 RNAiのクローンのライブラリの作成​​は 、Cの大部分をカバーしながら、 elegansのゲノム2,3（例4-7参照）真の機能ゲノム研究のために道を開いた、最も確立された方法は面倒です。モイらは、ゲノムワイドな画面8を容易にする半自動化されたプロトコルを開発しました。アプローチは、顕微鏡イメージングと画像解析に依存しています。 ここでは、細菌のRNAiのクローンのロボットハンドリング、COPAS Biosort（ユニオンBiometrica（UBI））を用いた定量分析などに基づいて、ハイスループットのゲノムワイドな画面の代替プロトコルを記述する統合ソフトウェア：MBioLIMS（MODULバイオからラボ情報管理システム）のデータ管理とサンプルを追跡するための高いスループットを実現する技術。メソッドは、画面が固形培地プレート上で実施することができます。これは、C言語でそれらのアドレッシング宿主-病原体相互作用などいくつかの研究のために特に重要です。 線虫は、特定の微生物を効率的に培養液でワームに感染していないので。 我々は、メソッドがCの抗真菌自然免疫の遺伝子の重要性を定量化するために使用することができる方法を示していますエレガンス 。このケースでは、このアプローチは、抗菌ペプチド遺伝子NLP 29とで構成的に発現される赤色蛍光レポーターのプロモーターの制御下にGFPを、表皮感染誘導蛍光レポーター遺伝子をトランスジェニック株の使用に依存しています表皮。後者は、表皮の機能の整合性が内部統制を提供していますと非特異的な遺伝子サイレンシング9。コントロールワームは真菌に感染しているとき、彼らは緑色蛍光を発する。 RNAiにより感染した後に減少した蛍光のNLP 29式の結果を得るために必要な遺伝子をノックダウン。現在、このプロトコルは、3,000以上のRNAiのクローンは2ヶ月以内にゲノム全体をスクリーニングの可能性を開いて、週ごとにテストし、分析することができます。

Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

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

我々は、使用してプロトコルを記述する<em&gt; C.エレガンス</em&gt;とRNAiはそのような集団内の個々のワームの蛍光、サイズや不透明度などの複数のパラメータの自動測定を可能にするライブラリを供給する。私達は中の抗真菌自然免疫に関与する遺伝子を識別するために画面の一例を与える<em&gt; C.エレガンス</em&gt;。

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. ...

quantitative-automated-high-throughput-genome-wide-rnai-screens-c

Research

JoVE Journal

Biology

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

17.6K ビュー.  Université de la Méditerranée. 我々は、使用してプロトコルを記述する C.エレガンスとRNAiはそのような集団内の個々のワームの蛍光、サイズや不透明度などの複数のパラメータの自動測定を可能にするライブラリを供給する。私達は中の抗真菌自然免疫に関与する遺伝子を識別するために画面の一例を与える C.エレガンス。

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

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

生物学

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

アイソフォーム特異的遺伝子標的を同定するためのチップを使用してゲノムワイド解析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

自動化を使用して染色体のハイスループット物理マッピング<em&gt;その場で</em&gt;ハイブリダイゼーション

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

ゲノムワイドな遺伝子欠失で<em&gt;ストレプトコッカス血統</em&gt;ハイスループットPCRによる

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegansの走化性アッセイ

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

内脂肪量の生化学的および高スループット微視的評価<em&gt;線虫（Caenorhabditis elegans）</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

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

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この動画の章

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

アイソフォーム特異的遺伝子標的を同定するためのチップを使用してゲノムワイド解析

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

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

自動化を使用して染色体のハイスループット物理マッピング

ゲノムワイドな遺伝子欠失で

C. elegansの走化性アッセイ

内脂肪量の生化学的および高スループット微視的評価

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

COSMICと細胞の薬剤-DNA相互作用のゲノムワイドなマッピング（小分子の架橋は、クロマチンを分離するために）