C. elegansの導入遺伝子は線虫遺伝学センター（ミネソタ大学、ミネアポリス、ミネソタ州）から提供された。この作品は、NIHのR21グラントKSへNS0667678 - 01によってサポートされていました。すべての著者は、収集、分析、およびデータの解釈にも参加。 CKL、KS、およびKPCは、原稿を書いたり、改訂に参加した。

1。細菌ワームの食品の調製日1 <ol><li>飽和E. 5 mlを加え大腸菌 OP50細菌培養に500ミリリットルのTerrificブロス50μg/ mlのストレプトマイシンを補充し、37℃で振盪インキュベーター（225 rpm）で一晩で成長℃の</li></ol>日2 <ol start="2"><li> 20分2,500 rcfで冷却遠心機で10 50mlチューブと遠心分離機の細菌に一晩細菌培養を分割する。 </li><li> LBブロスからデカントし、液体の線虫の増殖培地（NGM）の10mlにそれぞれの細菌のペレットを再懸濁します。細菌を懸濁するために15分間床シェーカーに水平に振る。 NGMバッファを作成するには、1リットルの脱イオン水とオートクレーブに3グラムのNaClを加える。 55℃まで冷却し、ために、以下の滅菌溶液を追加する：1 M CaCl 2で 、1 M MgSO 4を 1 mlの、そして1 Mリン酸カリウム、pH6.0の25mlのを1ml。 </li><li> 20分2,500 rcfで冷却遠心機で細菌培養を遠心分離します。 </li><li> NGMのバッファoffデカントし、細菌ペレットの重量を量る。 </li><li> 、ペレットを再懸濁させるNGM等量のバッファーを追加して細菌のアリコートを3ミリリットル、15 mlチューブに集中し、-20℃で保存します。 </li></ol> 2。大規模C. elegansの液体培養我々はトランスジェニックラインを使用VP596（dvIs19 [pAF15（GST - 4：：GFP：：NLS）]; vsIs33 [DOP - 3：：RFP]）は、2つの蛍光構造運ん：SKN - 1を監視するためにGFP 14：Pgst -4：活動とPDOP - 3：：RFP 17ワームの数の正規化のための標準として機能する。 日1 <ol><li> 150ミリリットルNGMバッファー、1.5 mlのLBブロス、5 mg / mlのコレステロールを150μl（エタノール）、および100 mg / mlストレプトマイシンの75μlを混合します。混合物をろ過滅菌し、1リットル無菌フラスコに加える。 注記：NGMバッファに1％のLBブロスを追加すると、ガラス器具やプラスチック容器に付着するのワームを防ぐのに役立ちますが、この量はバクテリアの成長をサポートするのに十分ではない。 </li><li>標準的な次亜塩素酸の手順18でワームを同期させます。まで妊娠ワームの0.5mlには次亜塩素酸溶液5ml（3.75ミリリットル滅菌水、1mlの家庭用漂白剤、および250μlの10NのNaOH）でシングル15 mlチューブで処理することができます。滅菌水でリリースされた卵を洗うとNGMの緩衝液10mlに再懸濁します。 </li><li>卵NGMバッファーで100倍（例えば、10ml中の100μlの）のサンプルを希釈し、3つの独立した5μlのアリコートで卵の数を数えます。卵の総数を推定するために20万の平均数（100希釈率× 2,000）、複数の。 </li><li> NGMのバッファ付きフラスコに20万〜200万の卵を追加し、20℃100rpmで振とう培養℃に</li></ol>日2 <ol start="5"><li>フラスコに卵を追加した後、少なくとも16時間、中断されたワームの文化の約0.5 mlを削除するには、無菌の血清学的ピペットを使用してください。ペトリ皿の滅菌蓋の上にピペットで3つの5μlの滴。ワームを麻痺させる1〜2分間-20℃の冷凍庫にふたをする。孵化したワームの総数を推定するために30,000（150ミリリットル/ 5μl）を、5μlとし、複数の当たりのライブ孵化ワームの平均数を数える。 </li><li>冷凍OP50細菌培養（議定書1.6）のチューブを融解、500,000斜線ワームあたりワームの文化に50パーセントOP50の3 mlを加える。 20℃で100rpmで振とうフラスコ℃をワームは、約51時間でL4幼虫や若い成人に発展する。細菌のOD600は、先頭に2.200程度にする必要があります。吸光度による細菌の量を監視し、OD 600が0.900を下回る場合は、よりを追加。 </li></ol> 3。ワームの収集及び調剤日4 <ol><li>標準NGM寒天培地にサスペンドワームの文化の約0.5 mlを転送するために滅菌血清ピペットを使用してください。ほとんどがL4幼虫や若い成人であることを確認するために実体顕微鏡でワームを見る。 L4幼虫は、体の真ん中で腹明確なスポットを持つことになります。若年成人は、わずかに大きく、明確なスポットを持っていないこととなります。 </li><li>滅菌50 mlのチューブにワームの文化を注ぎ、ベンチ上で試験管ラックにチューブを置きます。ワームは、約10分間静することができます。吸引またはピペットで上清を取り除きます。 注：この手順は、彼らはL4幼虫や若い成人よりも遅い解決するため、開発していない卵からかえっていない卵やワームを除去することが重要になることができます。 </li><li>単一の50 mlチューブに、すべてのワームを収集し、細菌を除去するために1％のLBブロス（NGM + LB）で3〜4回NGMバッファーで洗浄する。 注記：これは最高の30秒間に500 rcfで、ワームを収集し、新鮮なNGM + LBバッファーで上清を置き換えることにより、スイングローター遠心機で行われます。 </li><li>洗浄後、50mlに+ NGMとLBのバッファーをチューブに充填し、カスタムワーム調剤フラスコに注ぐ。小さな攪拌棒を追加し、ワームが一時停止を維持するためにかき混ぜる。目を数える上に示した3つの5μlの滴のワームの電子の数。あなたが5μlの降下（約2.5〜3.0ワーム/μL）あたり12月15日ワームを得るまでNGM + LBで希釈する。 注記：約20ml（60,000ワーム）の最小値は、調剤フラスコとディスペンサーカセットのデッドスペースを埋めるために必要とされる。各384ウェルプレートは、約35,000ワームやサスペンドワームの11.5 mlを必要とします。 </li><li> 10μlの調剤カセットをロードし、70％エタノールでプライミングによって滅菌する。滅菌水でプライミングによってエタノールをすすいでください。 </li><li>それだけでその動きを中断することなく、攪拌棒の上になるようにフラスコに分注カセットの端を差し込みます。ワームが全体フラスコを通して中断されたままにいることを確認してください。 2プリパルスで低速度で分配するようにプログラムディスペンサー。素数とは、一時停止ワームの少なくとも10 mlを実行します。チューブにセトリングからワームを防ぐために迅速に、必要に応じてプレートを埋める。 </li><li>通気性のテープでプレートをシールします。 </li><li>あなたのアッセイのための適切な温度でインキュベーター内で振とう台にプレートを置きます。プレートを積み重ねないでください。 </li></ol> 4。蛍光分析<ol><li>適切な発光と励起波長（：; RFP 540/25ex 590/35em GFP 485/20ex 528/20em私たちのアッセイのためのフィルタ）で各ウェルの蛍光強度を測定するためにマイクロプレートリーダーにターゲットプレートを置きます。 </li><li> GFP / RFPの比を計算し、個々の治療に由来する蛍光強度の正確な倍の差を決定するために対照ウェル（活性化化合物なし）の測定値で正規化する。 </li></ol> 5。代表的な結果： 私たちのSKN - 1アッセイは、染色体に統合されたデュアルレポーター株（VP596）を使用しています。図1に示すように、ワームの数は、よく分注されたボリュームに相関している。図2Aおよび2Bに示すように、ウェルあたりの総GFPとRFPの蛍光は十分によく384ウェルプレート全体から高い再現性です。などの非誘導Pgst - 4、図2Cに示すように：：GFPの蛍光は、直線的にPDOP - 3と相関している：：384ウェル間のRFP。 ：ばらつきを低減するためのRFPレポーター：GFP / RFPの比として表現したとき、蛍光はPDOP - 3の能力を示す384ウェルプレート（図2Dから2Aへの係数の変化を比較する）を介してウェルからウェルに高い再現性となる。図3に示すように、SKN - 1生体異物活性化（38μMjuglone）とRFPに対する相対的なGFPの誘導は、堅牢で384ウェルプレートで高い再現性です。 <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="図1"/> 図1。ボリュームに対するワームの数は、分注した。ワームは、約2/μlに希釈し、実体顕微鏡とマニュアルカウントのための24ウェルプレートに分注していた。体積当たりN = 8井戸。 <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="図2"/> 図2は、384ウェルプレートに分注されたワームの総蛍光は再現可能です。ワームは、約2.5/μl、30μlに希釈し、384ウェルプレートの各ウェルに分注した。すべてのウェルのGFP（A）とRFP（B）蛍光は、9％以下の変動係数を持っていた。 （C）GFP蛍光は非常にRFP蛍光相関している。 （D）RFPにGFPの比率を計算するには、下記の6％に変動係数を減少させた（A、B、およびD）実線は、手段を示し、破線は平均値の上または下にある3つの標準偏差を示している。 <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="図3"/> 。Pgst - 4の図3の活性化：：GFPは、堅牢で一貫性です。約75 L4ワームは、384ウェルプレートの全ウェルに分配され、38μMのjugloneは、他のすべての列に追加されました。 GFPとRFPの蛍光はインキュベーションの21時間後に測定した。すべてのコントロール（1.0）とjuglone井戸（8.9）の平均相対蛍光比を実線でマークされます。コントロール上の3つの標準偏差は意味とjuglone平均より破線でマークされます。

<ol>
	<li>Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-throughput+screen+identifies+inhibitors+of+the+Schistosoma+mansoni+redox+cascade.">Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.</a> PLoS Negl Trop Dis. 2, e127-e127 (2008).</li><li>Giacomotto, J., Segalat, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+small+animal+models,+where+are+we.">High-throughput screening and small animal models, where are we.</a> Br J Pharmacol. 160, 204-216 (2010).</li><li>Artal-Sanz, M., de Jong, L., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+a+versatile+platform+for+drug+discovery.">Caenorhabditis elegans: a versatile platform for drug discovery.</a> Biotechnol J. 1, 1405-1418 (2006).</li><li>An, J. H., Blackwell, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1+links+C.+elegans+mesendodermal+specification+to+a+conserved+oxidative+stress+response.">SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response.</a> Genes Dev. 17, 1882-1893 (2003).</li><li>Choe, K., Przybysz, A., Strange, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WD40+repeat+protein+WDR-23+functions+with+the+CUL4/DDB1+ubiquitin+ligase+to+regulate+nuclear+abundance+and+activity+of+SKN-1+in+Caenorhabditis+elegans.">The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans.</a> Mol Cell Biol. 29, 2704-2715 (2009).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide-responsive+genes+in+the+nematode+Caenorhabditis+elegans.">Acrylamide-responsive genes in the nematode Caenorhabditis elegans.</a> Toxicol. Sci. 101, 215-225 (2008).</li><li>Tullet, J. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+inhibition+of+the+longevity-promoting+factor+SKN-1+by+insulin-like+signaling+in+C.+elegans.">Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.</a> Cell. 132, 1025-1038 (2008).</li><li>Oliveira, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condition-adapted+stress+and+longevity+gene+regulation+by+Caenorhabditis+elegans+SKN-1/Nrf.">Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf.</a> Aging Cell. , (2009).</li><li>Park, S. -. K., Tedesco, P. M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+longevity+in+Caenorhabditis+elegans+as+mediated+by+SKN-1.">Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1.</a> Aging Cell. 8, 258-269 (2009).</li><li>Hayes, J. D., McMahon, M., Chowdhry, S., Dinkova-Kostova, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+chemoprevention+mechanisms+mediated+through+the+Keap1-Nrf2+pathway.">Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway.</a> Antioxid Redox Signal. , (2010).</li><li>Kwak, M. K., Kensler, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NRF2+signaling+for+cancer+chemoprevention.">Targeting NRF2 signaling for cancer chemoprevention.</a> Toxicol Appl Pharmacol. 244, 66-76 (2010).</li><li>Leiser, S. F., Miller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nrf2+signaling,+a+mechanism+for+cellular+stress+resistance+in+long-lived+mice.">Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice.</a> Mol Cell Biol. 30, 871-884 (2010).</li><li>Link, C. D., Johnson, 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=Reporter+transgenes+for+study+of+oxidant+stress+in+Caenorhabditis+elegans.">Reporter transgenes for study of oxidant stress in Caenorhabditis elegans.</a> Methods Enzymol. 353, 497-505 (2002).</li><li>Vanduyn, N., Settivari, R., Wong, G., Nass, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1/Nrf2+inhibits+dopamine+neuron+degeneration+in+a+Caenorhabditis+elegans+model+of+methylmercury+toxicity.">SKN-1/Nrf2 inhibits dopamine neuron degeneration in a Caenorhabditis elegans model of methylmercury toxicity.</a> Toxicol Sci. , (2010).</li><li>Hasegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+inexpensive+method+to+screen+for+common+foods+that+reduce+the+action+of+acrylamide,+a+harmful+substance+in+food.">A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food.</a> Toxicol Lett. 175, 82-88 (2007).</li><li>Chase, D. L., Pepper, J. S., Koelle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+extrasynaptic+dopamine+signaling+in+Caenorhabditis+elegans.">Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans.</a> Nature Neuroscience. 7, 1096-1103 (2004).</li><li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans, a Practical Guide. , (2005).</li><li>Pulak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+analysis,+sorting,+and+dispensing+of+C.+elegans+on+the+COPAS+flow-sorting+system.">Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system.</a> Methods Mol Biol. 351, 275-286 (2006).</li></ol>

利害の衝突は宣言されません。

我々は、培養する方法を提示し、トランスジェニック線虫の多数を調剤。培養のワームに使用される機器は、分子のクローニングを行う検査室の標準であり、液体のハンドリングと蛍光装置は、マイクロプレートを大量に処理する研究室の標準です。ライブC.多数の調剤の他の方法虫は、機器19をソートする高価な粒子が必要です。 Pgst - 4：：GFPアッセイは、キャ?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> LBブロス</td><td>研究プロダクツインターナショナル株式会社</td><td> L24066 </td><td></td></tr><tr><td>素晴らしいスープ</td><td>研究プロダクツインターナショナル株式会社</td><td> T15100 </td><td></td></tr><tr><td>シナジー™HTマルチモードマイクロプレートリーダー</td><td> BioTekの</td><td></td><td>フィルター：GFP 485/20ex 528/20em、RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo選択ディスペンサー</td><td> BioTekの</td><td></td><td></td></tr><tr><td>ワーム調剤フラスコ</td><td>南サイエンティフィック社、Micanopy、フロリダ州</td><td></td><td>カスタムは（352）284から2531を組み立てました</td></tr><tr><td> 384マイクロプレート</td><td>グライナーバイオワン</td><td> 5678-1209 </td><td></td></tr><tr><td>通気性のシールテープ</td><td>ヌンク</td><td> 241205 </td><td></td></tr><tr><td> （5 - ヒドロキシ- P - naphthoqinone）Juglone </td><td>アクロス</td><td> 121640010 </td><td> JugloneはDMSOに溶解し、 </td></tr><tr><td> DMSO </td><td>シグマアルドリッチ</td><td> D - 5879 </td><td></td></tr><tr><td> C. elegansのトランスジェニック系統</td><td>著者の研究室</td><td> VP596 </td><td> Pgst - 4：：GFPとPDOP - 3：：RFP </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

ハイスループットスクリーニング（HTS）は、生物学的プロセスの化学的モジュレーターを同定するための強力なアプローチです。しかし、細胞培養モデルを使用して画面で識別される多くの化合物はしばしば生体 1-2 の毒性または薬理学的に不活性であることがわかっている。全体の動物モデルでのスクリーニングは、これらの落とし穴を回避し、薬剤開発へのパスを効率化できます。 C. elegansはよくHTSに適した多細胞モデル生物です。それは小さい（&lt;1 mm）と経済的に培養し、液体中で省略することができる。C.は虫はまた、薬物モードのアクション3の迅速かつ詳細な識別を可能にする最も実験的に扱いやすい動物モデルの一つです。 我々は、培養のためのプロトコルを記述し、Cの蛍光菌株を分注する化合物ライブラリーまたは特定の遺伝子の発現を変化させる環境汚染物質の検出のハイスループットスクリーニングのためのエレガンス 。発達同期ワームの多数は、液体培養で増殖収穫、洗浄、および定義された密度で懸濁する。ワームはその後、蠕動液体ディスペンサーを使用して黒、平底の384ウェルプレートに追加されます。化学物質のライブラリまたは試験サンプル（例えば、水、食物、または土壌）からの小分子は、ワームとのウェルに添加することができます。 生体内では 、リアルタイムの蛍光強度を蛍光マイクロプレートリーダーで測定される。このメソッドは 、Cの任意の誘導性遺伝子に適合させることができます適切なレポーターが使用可能なエレガンス 。多くの誘導性ストレスと発達転写経路はよくCで定義されています。 虫とGFPトランスジェニックレポーター株は、すでにその4の多くのために存在する。適切なトランスジェニック記者と組み合わせることで、私たちの手法は、経路の調節因子をスクリーニングするか、環境汚染物質のための堅牢なバイオアッセイを開発するために使用することができます。 私たちは、Cを示しています虫の文化と我々はCを監視するために開発されたHTSアッセイと調剤プロトコル虫キャップ&#39;N&#39;カラー転写因子SKN - 1。 SKN - 1とその哺乳類ホモログのNRF2は5-10酸化や生体異物のストレス時の細胞保護遺伝子を活性化する。 NRF2は、がん、神経変性疾患、および慢性炎症のような多数の加齢に伴う障害から哺乳類を保護し、主要な化学療法の標的11から13になっています。私たちのアッセイを-4 SKN - 1の標的遺伝子GST 14のGFPトランスジェニックレポーターに基づいて、これは、グルタチオン- Sトランスフェラーゼ6をエンコードします。 GST -4記者はまた、SKN - 1を活性化し、そのようなアクリルアミドとメチル水銀15から16のような汚染物質の低レベルを検出するために使用できる生体異物と酸化的化学物質のバイオセンサーです。

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

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

液体ベースの培養および調剤のための手順<em&gt; C.エレガンス</em蛍光レポータータンパク質を発現する&gt;株は、高価な仕分け装置を必要としないことが記載されている。このアプローチは、多数の誘導に適用することができます<em&gt; C.エレガンス</em&gt;汚染物質の創薬やバイオセンシングのための遺伝子。

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 ...

high-throughput-screening-biosensing-with-fluorescent-c-elegans

Research

JoVE Journal

Biology

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

17.9K ビュー.  University of Florida. 液体ベースの培養および調剤のための手順 C.エレガンス株は、高価な仕分け装置を必要としないことが記載されている。このアプローチは、多数の誘導に適用することができます C.エレガンス汚染物質の創薬やバイオセンシングのための遺伝子。

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

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

の生産 C.エレガンス</emとRecombineering経由>トランスジーン galK選択可能なマーカー

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

アフィニティータグ組換えタンパク質のハイスループット精製

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向き整流カリウムチャネルの小分子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

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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 Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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 ...

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

要約

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の生産 C.エレガンストランスジーン galK選択可能なマーカー

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌

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

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

アフィニティータグ組換えタンパク質のハイスループット精製

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