この作品は、受賞数R01HG006785下の国立衛生研究所の国立ヒトゲノム研究所によってサポートされていました。

1配列設計と合成<ol><li>調節活性についてアッセイすべき配列の設計と合成にMPRAを開始します。 pMPRAベクターシリーズとの互換性のために、次のテンプレートを使用して、各シーケンスを設計する：5&#39;-ACTGGCCGCTTCACTG-のVAR -GGTACCTCTAGA- タグ -AGATCGGAAGAGCGTCG-3 varはアッセイすべき配列を意味し、 タグは 、1つまたは複数の識別タグを表す&#39;。 </li><li> 2つの可変領域（varとタグ ） は 、それらの間のレポーター遺伝子断片の方向性ライゲーションを容易にするためにKpnI（GGTACC）およびXbaI（TCTAGA）制限部位の対によって分離されている。さらに、2つの別個のSfiI（GGCCNNNNNGGCC）部位はpMPRA骨格に指向性ライゲーションを可能にするPCRによって追加される。可変領域は、これらの制限部位のいずれかの追加のコピーを含んではならない。必要であれば、これらの制限酵素の1つ以上が置換され得る限り代替品として1）に近いDNA分子の末端への効率的な切断を可能にし、2）ベクター配列の他の場所で切断しない。追加の詳細についての説明を参照してください。 </li><li>商用ベンダーから、表1にリストされている必要なプライマーを入手します。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="381"><tbody><tr><td> MPRA_SfiI_F </td><td> GCTAAGGGCCTAACTGGCCGCTTCACTG </td></tr><tr><td> MPRA_SfiI_R </td><td> GTTTAAGGCCTCCGTGGCCGACGCTCTTC </td></tr><tr><td> TAGseq_P1 </td><td> AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGACGCTCTTCCGATCT </td></tr><tr><td> TAGseq_P2 </td><td> CAAGCAGAAGACGGCATACGAGAT [インデックス] GT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGG </td></tr></tbody></table> 表1プライマー配列。[インデックス]多重化された配列決定のために使用される6〜8ヌクレオチドのインデックスシーケンスを示している。異なる屈折率を有する少なくとも8 TagSeq-P2プライマーを取得します。すべてプライマーのHPLCまたはPAGEによって精製されるべきである。 <ol start="4"><li>オリゴヌクレオチドライブラリーは、商業ベンダーまたはコア施設から得られた、または製造者によって記載されるようにプログラム可能なマイクロアレイに基づくオリゴヌクレオチド合成機を用いて生成することができる。 100μlのTE 0.1バッファにOLSの製品を再懸濁します。 </li><li> 10％TBE-尿素変性ポリアクリルアミドゲル上のオリゴヌクレオチドライブラリーを実行します。その品質（ 図2A）を評価するのssDNA感受性蛍光染色でレーンや汚れ当たり約1ピコモルをロードします。 </li><li>完全長のオリゴヌクレオチドに対応する離散的なバンドが可視化することができる場合（ 図2A、レーン1および2）、対応するアクリルアミドゲルスライスを切り出し、振盪しながら室温で一晩、100μlのTE 0.1にオリゴヌクレオチドを溶出する。そうでなければ、生のOLS懸濁液を用い進みます。 </li><li>エマルジ ​​ョンPCR 9を使用して、オリゴヌクレオチドライブラリーへのSfiI尾を増幅し、追加</<html> SUP&gt;。 </li><li> 表2に記載したように、50μlのPCR反応混合物をセットアップします。その後、4℃で5分間、300μlのMicellula DNAエマルジ ​​ョンからの油と界面活性剤の混合物および精製キットおよび渦と組み合わせる。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="368"><tbody><tr><td>試薬</td><td> 1倍容量（μL） </td></tr><tr><td>ヘラクレスII融合DNAポリメラーゼ</td><td> 0.5 </td></tr><tr><td>ヘラクレスII反応緩衝液を5倍</td><td> 10 </td></tr><tr><td>のdNTP（それぞれ10mM） </td><td> 1.25 </td></tr><tr><td> BSA（20 mg / ml）を</td><td> 1.25 </td></tr><tr><td>プライマーMPRA_SfiI_F（25μM） </td><td> 0.25 </td></tr><tr><td>プライマーMPRA_SfiI_R（25μM） </td><td> 0.25 </td></tr><tr><td> OLSテンプレート（1-10 attomol） </td><td>変わる</td></tr><tr><td>ヌクレアーゼフリー水</td><td> 50 </td></tr></tbody></table>ve_content &quot;&gt; 表2エマルジ ​​ョンPCR反応混合物（水相）。 <ol start="9"><li>アーティファクトの出現なしに増幅産物の最大収量が得られる濃度を使用してください（セクション1.12を参照）。 </li><li> PCRチューブに得られたエマルジョンを分配し、95℃（2分20サイクルのPCRを行い、20×[95℃で20秒、55°C、72°Cで30秒、20秒]、95℃で3分間°C）。 </li><li>プールDNA精製カラムを用いて増幅産物を精製し、次いでイソブタノールを1ml添加​​し、短時間ボルテックスし、そしてにより各350μlのエマルジョンPCR反応を破壊する。 </li><li>アーチファクトのない増幅（ 図2B）を確認するために、2％または4％アガロースゲル上でアリコートを実行する。 </li></ol><img alt="図2" src="/files/ftp_upload/51719/51719fig2highres.jpg" /> オリゴヌクレオチド合成ライブラリーの調製図。A） </s·チョン&gt;三つの異なる生のオリゴヌクレオチド合成ライブラリ（OLS）は、10％TBE-尿素変性ポリアクリルアミドゲル上で実行されます。完全長オリゴヌクレオチド（*）に対応するバンドを可視化し、ライブラリ1から切り出し、2ライブラリ3は、PAGE精製を妨害する汚染物質が含まれていることができます。この場合は、アガロースゲル上で同一のオリゴヌクレオチドライブラリーのランの開放およびエマルジ ​​ョンPCR増幅のPCR増幅。B）製品に直接進む。複雑なオリゴヌクレオチドライブラリーのPCR増幅は、頻繁に上位と下位のバンドとして表示されることがあり、キメラ製品、その他の成果物を作成します。エマルジョンPCRは、これらの成果物を最小限に抑えることができます。 2ライブラリー構築<ol><li> 1％アガロースゲル上で泳動し、2時間、50℃でのSfiIを有するベクターpMPRA1を消化することによって線状化プラスミドバックボーンを準備し、（この場合、2.5キロバイト）をバックボーンバンドを切り出し、ゲル精製スピンカラムを用いて精製する。 </li><li>ダイジェスト2時間、50℃でのSfiIとステップ1.4からのエマルジ ​​ョンPCR産物およびDNA精製カラムを用いて精製する。 </li><li> 、線状化ベクターバックボーンにプロモーター - タグライブラリを連結するために消化したPCR産物100ngの、直線化ベクター骨格およびT4 DNAリガーゼ50ngのを含む反応を設定します。 16℃で一晩インキュベートし、次にリガーゼを不活性化するために20分間65℃で加熱する。 </li><li> E.トランスフォームライゲーション反応で大腸菌 。ライブラリの複雑さを維持するために、設計されたライブラリ内の個別のプロモーター - タグ組み合わせが存在するよりも、少なくとも10倍以上のCFUを得ることを目指しています。 </li><li>タグの合計数​​は約10万である場合、ターゲットのcfuは、典型的には、ライブラリごと6-8パラレル変換を実行することによって達成することができる。 </li><li>各形質転換のために、50μlのエレクトロコンピを組み合わせる氷上の連結混合物2μlを持つ大腸菌細胞 。エレクトロポレーションによりトランスフォーム、800μlのSOCの細胞を回収1時間37℃で振とうによって培地、その後、パラレル変換から細胞を兼ね備えています。 </li><li> 50〜100 / mlのカルベニシリンを含むLB寒天プレート上で回収された細胞のalinquotから、プレート連続希釈を形質転換効率を評価し、37℃で一晩インキュベートする。 / V、Vが回収された細胞とvの全容積で連続希釈に要した一定分量の体積である-総CFUとして（観測CFU）×（希釈倍率）×（V V）を推定する。 </li><li>同時に、回収された細胞の残りは100μgの/ mlのカルベニシリンを補充した200ミリリットルのLBに接種するために使用する。シェーカーインキュベーター内で37℃で一晩、細胞を成長させ、その後、標準的な手順に従って、プラスミドDNAを単離する。 </li><li> 2時間50℃でのSfiIを有する単離プラスミドライブラリーのアリコートを消化し ​​、インサートの存在を確認するために1％アガロースゲル上で泳動。 </li><li> cでプラスミドライブラリーを直線化KpnI及びXbaIでを使用して、プロモーター変異体とタグの間にウッティング。消化効率を最大化するために、シリアル消化を実行します。 </li><li>まず、1時間、37℃でKpnIで消化し ​​た磁気ビーズを用い精製する。第二に、2時間、37℃で1 Uエビアルカリホスファターゼを添加したXbaIで消化、熱は、5分間65℃で不活性化し、その後、磁気ビーズを用いて精製する。 </li><li>完全な線形化を確認するために1％アガロースゲル上でアリコートを実行する。ノーカットプラスミドが表示されている場合は、線形化された断片をゲル精製しなければならない。 </li><li>哺乳動物細胞へのトランスフェクションに適しMPRAライブラリを生成するには、線形化された中間ライブラリにのKpnI / XbaIで互換の端部とのORFを連結。 </li><li> pMPRAdonor1から互換luc2 ORF断片を調製し、1時間37℃でKpnI及びXbaIでこのプラスミドを消化し ​​、1％アガロースゲル上で泳動。 ORFのフラグメントを切り出し（1.7 Kこの場合、b）およびゲル精製スピンカラムを用いて精製する。 </li><li>ステップ2.3から2.8に記載されているように線形化された中間ライブラリにORFのフラグメントをクローニングします。 </li><li> 1時間、37℃でKpnIでMPRAライブラリ（1-2μgのに対応）分量を消化し ​​、1％アガロースゲル上で実行されます。消化されたライブラリが単一のバンドとして実行する場合、追加のバンドが認められた場合には、（通常は中間の構築物のキャリーオーバーに相当）のセクション3に進んで次のように、ライブラリは、さらに精製することができる。 </li><li> 1時間、37℃でKpnIで汚染されたライブラリの3-5μgのを消化し ​​、4℃で一晩0.8％アガロースゲル上で実行されます。 </li><li> 、正しいライブラリバンドを切り出し、ゲル精製スピンカラムを用いてDNAを精製し、1時間37℃で高濃度T4 DNAリガーゼを使用してセルフライゲーションを行う。ステップ2.8で説明したようにその後転換および最終ライブラリーDNAの単離を繰り返します。 </li></ol> 3 Transfection、摂動、およびRNAの単離<ol><li>それぞれ独立したトランスフェクション、文化、適切な培地中の細胞に必要な数（MPRAライブラリの複雑さによって決定されるように、ディスカッションを参照）。例えば、DMEM中で培養HEK293T / 17細胞を、10％FBSおよびL-グルタミン/ペニシリン/ストレプトマイシンを補充した。各ライブラリおよび実験条件について、少なくとも2つの独立したトランスフェクションのための培養細胞。 </li><li> MPRAプラスミドを用いて培養細胞をトランスフェクション。トランスフェクション方法および条件は、各細胞型について最適化されなければならない。各トランスフェクトサンプルについて、一致させた対照として、プラスミドDNAの一部（50〜100 ngの）を保持。 </li><li>例えば、1ミリリットルのOpti-MEM中10μgのプラスミドDNAを用いた10cmの培養皿内の約50％コンフルエンスまで増殖させた0.5×10 7 HEK293T / 17細胞をトランス私は30μlのリポフェクタミンLTXと10μlのPLUS試薬を使用した低血清培地。 5時間後、トランスフェクション混合物を削除し、許可する細胞は24〜48時間回復する。 </li><li>必要に応じて、設計されたライブラリ内の文脈や信号依存調節配列を活性化するために必要な摂動を行う。 </li><li>細胞を採取し、そのメーカーの指示を使用して、標準的なオリゴ（dT）セルロースカラムやビーズを使用してポリ（A）+ mRNAを単離する。 </li><li>カラムやビーズの最大結合容量は採取された細胞から期待するmRNAの総量を超えていることを確認してください。例えば、セクション3.3から予想される収量は約0.5〜2.5μgのmRNAである。 </li></ol> 4。タグ配列<ol><li>トランスフェクトされた細胞溶解物からベクターDNAのキャリーオーバーをなくすために、で2.4μlのターボDNアーゼ不活性化試薬を、1時間、37℃で1μlのターボのDNase（2U）と、2.3μlの10倍のターボのDNaseバッファーで各20μlのmRNA試料を扱う追加その後、混合、90秒10,000 gで遠心分離し、新しいチューブにソリューションを移すとの5分間のRT。 </li><li>確認する<html>各mRNAサンプルの60〜100 ngの上のセクション4.6で説明した後、アガロースゲル上で製品を実行するようにしてPCRを行うことにより、純度。 （luc2でpMPRA1を使用している場合は0.25キロバイト）特定のアンプリコンが表示されている場合、その列は、処理されたmRNAを精製した後、DNase処理を繰り返します。 </li><li> 、タグ配列のシーケンシングライブラリーを作製cDNAにレポーターmRNAを変換して、PCRによる配列決定アダプターを追加します。 </li><li> 表3に記載したようにmRNAを/ RTプライマー混合物をセットアップします。その後、氷上に置き、5分間65℃でインキュベートする。 表4に記載したように並行して、cDNA合成反応をセットアップする。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td>試薬</td><td> 1倍容量（μL） </td></tr><tr><td> mRNAサンプル（400〜700 ngの合計） </td><td> 8 </td></tr><tr><td>オリゴ0dT（50μM） </td><td> 1 </td></tr><tr><td>のdNTP（それぞれ10mM） </td><td> 1 </td></tr></tbody></table>cDNA合成のため ove_content &quot;&gt; 表3 RNA /逆転写プライマーミックス。 <table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td>試薬</td><td> 1倍容量（μL） </td></tr><tr><td> 10倍のスーパースクリプトIII RTバッファー</td><td> 2 </td></tr><tr><td>のMgCl 2（25mMの） </td><td> 4 </td></tr><tr><td> DTT（0.1M） </td><td> 2 </td></tr><tr><td> RNaseOut（40 U /μl）を</td><td> 1 </td></tr><tr><td>スーパースクリプトIII（200 U /μl）を</td><td> 1 </td></tr></tbody></table> 表4 cDNA合成反応混合物。 <ol start="5"><li>ゆっくりするmRNA / RTプライマーがcDNA合成ミックスと混合兼ね備えています。 50分、5分、85℃、50℃でインキュベートした後、氷上に置きます。最後に、20分間37℃で2 UリボヌクレアーゼのHでインキュベートする。 </li><li> 4-6μlの表5に記載したようにPCR反応をセットアップしますcDNA反応混合物または鋳型として50 ngのレポータープラスミド。そして（3分間、72°C [30秒間、30秒間、30秒間95°C 72°Cで55°C]を2分間、26 xに対して95°C）PCR増幅を行う。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td>試薬</td><td> 1倍容量（μL） </td></tr><tr><td> PfuUltra II HotStartのPCRマスターミックスの2倍</td><td> 25 </td></tr><tr><td>プライマーTagSeq_P1（25μM） </td><td> 0.5 </td></tr><tr><td>プライマーTagSeq_P2（25μM） </td><td> 0.5 </td></tr><tr><td>テンプレート（mRNAは、cDNAの混合物またはプラスミドDNA） </td><td>変わる</td></tr><tr><td>ヌクレアーゼフリー水</td><td> 50 </td></tr></tbody></table> 表5タグ配列のPCR反応ミックス。 <ol start="7"><li> 2％アガロースゲルでPCR産物を実行し、タグ配列のライブラリアンプリコンに対応する物品のバンド（0.25キロバイト使用している場合luc2とpMPRA1）、これらを用いてゲル精製スピンカラムを精製する。 </li><li>プール、イルミナシーケンシング装置と直接、精製タグ配列アンプリコンを変性させ、配列決定する。 </li><li>低品質a）はシーケンスされたタグ内に30未満のスコアまたはb）正確に設計されたタグと一致していないのPhred品質で一つ以上の位置が含まれているすべてのことを削除することで読み込みフィルター。残りの各タグは、各ライブラリに表示された回数をカウントします。 TPMに（百万配列を決定したタグごとにタグ）タグ数を正規化した後、シーケンシングライブラリの各ペアのためのプラスミド由来のタグカウントオーバーのmRNA由来のタグカウントの比を計算する。複数の異なるタグが各配列変異体に結合させた場合には、下流の解析のために彼らの中央値の比率を使用しています。 </li></ol>

<ol>
	<li>Kinney, J., Murugan, A., Callan, C. G., Cox, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+deep+sequencing+to+characterize+the+biophysical+mechanism+of+a+transcriptional+regulatory+sequence.">Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence.</a> Proceedings of the National Academy of Sciences USA. 107 (20), 9158-9163 (2010).</li><li>Melnikov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+dissection+and+optimization+of+inducible+enhancers+in+human+cells+using+a+massively+parallel+reporter+assay.">Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay.</a> Nature Biotechnology. 30 (3), 271-277 (2012).</li><li>Patwardhan, R. P., Lee, C., Litvin, O., Young, D. L., Pe&#8217;er, D., Shendure, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+analysis+of+DNA+regulatory+elements+by+synthetic+saturation+mutagenesis.">High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis.</a> Nature Biotechnology. 27 (12), 1173-1175 (2009).</li><li>Patwardhan, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+functional+dissection+of+mammalian+enhancers+in+vivo.">Massively parallel functional dissection of mammalian enhancers in vivo.</a> Nature Biotechnology. 30 (3), 265-270 (2012).</li><li>Arnold, C. D., Gerlach, D., Stelzer, C., Bory&#324;, &. #. 3. 2. 1. ;. M., Rath, M., Stark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+quantitative+enhancer+activity+maps+identified+by+STARR-seq.">Genome-wide quantitative enhancer activity maps identified by STARR-seq.</a> Science (New York, N. Y.). 339 (6123), 1074-1077 (2013).</li><li>Kheradpour, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+dissection+of+regulatory+motifs+in+2000+predicted+human+enhancers+using+a+massively+parallel+reporter+assay.">Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay.</a> Genome Research. 23 (5), 800-811 (2013).</li><li>White, M. A., Myers, C. A., Corbo, J. C., Cohen, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+in+vivo+enhancer+assay+reveals+that+highly+local+features+determine+the+cis+-regulatory+function+of+ChIP-seq+peaks.">Massively parallel in vivo enhancer assay reveals that highly local features determine the cis -regulatory function of ChIP-seq peaks.</a> Proceedings of the National Academy of Sciences USA. 110 (29), 11952-11957 (2013).</li><li>Mogno, I., Kwasnieski, J. C., Cohen, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+synthetic+promoter+assays+reveal+the+in+vivo+effects+of+binding+site+variants.">Massively parallel synthetic promoter assays reveal the in vivo effects of binding site variants.</a> Genome Research. 23 (11), 1908-1915 (2013).</li><li>Sch&#252;tze, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+emulsion+polymerase+chain+reaction+and+subsequent+purification.">A streamlined protocol for emulsion polymerase chain reaction and subsequent purification.</a> Analytical Biochemistry. 410 (1), 155-157 (2011).</li><li>Panne, D., Maniatis, T., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+atomic+model+of+the+interferon-beta+enhanceosome.">An atomic model of the interferon-beta enhanceosome.</a> Cell. 129 (6), 1111-1123 (2007).</li></ol>

著者は、彼らが競合する金融利害がないことを宣言します。

MPRA is a flexible and powerful tool for dissection of sequence-activity relationships in gene regulatory elements. The success of MPRA experiments depend on at least three factors: 1) careful design of the sequence library, 2) minimization of artifacts during amplification and cloning, and 3) high transfection efficiency. 
The possible lengths of the variable regions in the reporter constructs are largely determined by the synthesis or cloning technology used. Standard OLS is generally limited to about 200 nt, but this protocol is compatible with inserts up to at least 1,000 nt. Note that variable regions that are highly repetitive or contain strong secondary structures may end up underrepresented due to PCR and cloning biases. The length of the tags that identify each of the variable regions should be 10-20 nt and the collection of tags should ideally be designed such there are at least two nucleotide differences between any pair. Tags that contain the seed sequences of known microRNA or other factors that might influence mRNA stability should also be avoided when possible.
A key parameter in the design of MPRA experiments is the total number of distinct reporter constructs to be included in the library (the design complexity, denoted CD). In practice, CD is limited by the number of cultured cells that can be transfected. As a rule of thumb, the total number of transfected cells should be at least 50-100 times greater than CD. For example, if 20 million cells can be transfected with a transfection efficiency of 50%, then CD should be no more than ~200,000. Note that CD is equal to the number of distinct regulatory sequence variants multiplied by the number of distinct tags per sequence. The more distinct tags are linked to each regulatory sequence, the more accurate the estimate of the activity of that sequence can be made (because measurements from distinct tags can be averaged), but the fewer distinct variants can be assayed in one experiment. The optimal choice depends on the experimental design. In a simple &#8220;promoter bashing&#8221; experiment, where a mathematical model will be fitted to the aggregated measurements, a single tag per variant is usually sufficient. In a screen for single-nucleotide polymorphisms that cause changes in regulatory activities, it may be necessary to use 20 or more tags per allele to obtain statistically robust results, because comparing each pair of alleles requires a separate hypothesis test. 
If the sequences to be assayed are not expected to contain transcription start sites, a constant promoter can also be added in the same fragment. For example, pMPRAdonor2 (Addgene ID 49353) includes a minimal TATA-box promoter that is useful when the upstream variable region is expected to have significant enhancer activity, while pMPRAdonor3 (Addgene ID 49354) includes a modified, strong SV40 viral promoter that is useful when the variable region is expected to contain silencer activity or other negative regulatory elements. 
Raw OLS products often contain a significant fraction of truncated oligonucleotides. These may interfere with accurate PCR amplification of the designed sequences, particularly when there is significant homology between them. Using PAGE purification to remove truncated synthesis products and emulsion PCR to minimize amplification artifacts are effective techniques for ensuring high library quality. If either step is impractical, it is imperative to minimize the number of PCR cycles used at each amplification step. Selection and expansion of the cloned library in liquid culture is generally sufficient to maintain the design complexity, but if recombination-prone vectors are to be used or significant representation bias is observed, the recovered cells can instead be plated directly onto large LB agar plates, expanded as individual colonies and then scraped off for DNA isolation. It is also important to consider the potential impact of synthesis errors, which are typically found at a rate of 1:100-500 in OLS. Full-length sequencing of the reporter constructs prior to transfection is recommended to identify and correct for such errors.
It is not necessary to introduce reporter constructs into every cell in the transfected culture, but transfection efficiencies below ~50% may lead to poor signal to noise ratios. It is advisable to optimize transfection conditions prior to performing MPRA experiments in a new cell type. When working with hard-to-transfect cell types, MPRA signals can be boosted by pre-selecting transfected cells. The pMPRA vector series includes variants that constitutively express a truncated cell surface marker that can be used to physically enrich for transfected cells prior to RNA isolation (for example, Addgene IDs 49350 and 49351).

A correction to the author list has been made for article <a href="http://www.jove.com/video/51719">Massively Parallel Reporter Assays in Cultured Mammalian Cells</a>.

Erratum: Massively Parallel Reporter Assays in Cultured Mammalian Cells

超並列レポーターアッセイ（MPRA）DNA配列1〜7の数十万人に何千もの転写調節活動の多重化された測定を可能にする。その最も一般的な実装では、多重化は、オープンリーディングフレーム（; 図1 ORF）の下流に識別するシーケンスタグを含む合成レポーター遺伝子に関心のある各シーケンスを結合することによって達成される。トランスフェクション、RNA単離およびレポーター遺伝子転写物の3 &#39;末端の深いシーケンシングの後、結合された配列の相対的活性は、それらの識別タグの相対存在量から推測することができる。 <img alt="図1" src="/files/ftp_upload/51719/51719fig1highres.jpg" /> MPRAの図1。概要。MPRAレポーター構築物のライブラリーは、合成に推定される調節配列に結合することによって構築される（例えばGFP又はルシフェラーゼのような）「不活性」、ORFから成るレポーター遺伝子は、識別配列タグが続く。ライブラリーは、培養細胞の集団に一斉にトランスフェクトされ、転写レポーターmRNAはその後回収される。ディープシークエンシングは、レポーターmRNAおよびトランスフェクトプラスミドの間で、各タグの出現回数をカウントするために使用される。プラスミドカウント上のmRNAのカウントの比率が、対応する調節配列の活性を推測するために使用することができる。メルニコフの許可を得て適応さら 2。 MPRAは、対象の遺伝子座を横切っ新規調節エ​​レメントに対する個別の遺伝子調節要素の1）総合的突然変異誘発、2）スキャンを含む、実験デザインの多種多様に適合させることができる、3）推定上の一組の天然の遺伝的変異の効果を試験するプロモーター、エンハンサーまたはサイレンサー、合成調節要素の4）の半合理的なエンジニアリング。リー配列変異体のbrariesは、プログラマブルマイクロアレイ2,3,6,7、縮重オリゴヌクレオチド1,4のアッセンブリー、コンビナトリアルライゲーション8およびゲノムDNA 5の断片化に対するオリゴヌクレオチドライブラリー合成（OLS）を含む、さまざまな方法を用いて生成することができる。 （それぞれAddgene IDが49349と49352、; http://www.addgene.org）このプロトコルはOLSとpMPRA1とpMPRAdonor1ベクターを用いて、変異型プロモーターのライブラリーの構築について説明し、培養された哺乳動物細胞へのこのライブラリの一過性トランスフェクションおよびその後の定量それらに関連するタグ（タグ配列）の深い配列決定によるプロモーター活性の。このプロトコルの以前のバージョンでは、（2013）研究23、800から811メルニコフらネイチャー·バイオテクノロジー30、271-277（2012）およびKheradpour らゲノムに報告され、研究で使用した。

<table><tbody><tr><td>Oligonucleotide library synthesis</td><td>Agilent, CustomArray, or other OLS vendors</td><td>custom</td><td>If using OLS construction method</td></tr><tr><td>pMPRA1</td><td>Addgene</td><td>49349</td><td>MPRA plasmid backbone</td></tr><tr><td>pMPRAdonor1</td><td>Addgene</td><td>49352</td><td>luc2 ORF donor plasmid</td></tr><tr><td>TE 0.1 Buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)</td><td>Generic</td><td>n/a</td><td>OLS buffer</td></tr><tr><td>Novex TBE-Urea Gels, 10%</td><td>Life Technologies</td><td>EC6875BOX</td><td>PAGE purification of OLS products</td></tr><tr><td>Novex TBA-Urea Sample Buffer</td><td>Life Technologies</td><td>LC6876</td><td>PAGE purification of OLS products</td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Life Technologies</td><td>S-11494</td><td>PAGE purification of OLS products</td></tr><tr><td>Micellula DNA Emulsion &amp;amp; Purification Kit</td><td>EURx/CHIMERx</td><td>3600-01</td><td>Library amplification by emulsion PCR</td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent</td><td>600675</td><td>Polymerase for emulsion PCR</td></tr><tr><td>SfiI</td><td>New England Biolabs</td><td>R0123S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>KpnI-HF</td><td>New England Biolabs</td><td>R3142S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>XbaI</td><td>New England Biolabs</td><td>R0145S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>T4 DNA Ligase (2,000,000 units/ml)</td><td>New England Biolabs</td><td>M0202T</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>One Shot TOP10 Electrocomp &lt;em&gt;E. coli&lt;/em&gt;</td><td>Life Technologies</td><td>C4040-50</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>LB agar and liquid media with carbenicllin</td><td>Generic</td><td>n/a</td><td>Growth media for cloning</td></tr><tr><td>E-Gel EX Gels 1%</td><td>Life Technologies</td><td>G4010-01</td><td>Library verification and purification</td></tr><tr><td>E-Gel EX Gels, 2%</td><td>Life Technologies</td><td>G4010-02</td><td>Library verification and purification</td></tr><tr><td>MinElute Gel Extraction Kit</td><td>Qiagen</td><td>28604</td><td>Library and backbone purification</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Library DNA isolation</td></tr><tr><td>Cell culture media</td><td>n/a</td><td>n/a</td><td>Experiment-specific</td></tr><tr><td>Transfection reagents</td><td>n/a</td><td>n/a</td><td>Experiment-specific</td></tr><tr><td>MicroPoly(A)Purist Kit</td><td>Life Technologies</td><td>AM1919</td><td>mRNA isolation</td></tr><tr><td>TURBO DNA-free Kit</td><td>Life Technologies</td><td>AM1907</td><td>Plasmid DNA removal</td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Life Technologies</td><td>18080-051</td><td>cDNA synthesis</td></tr><tr><td>PfuUltra II Hotstart PCR Master Mix</td><td>Agilent</td><td>600850</td><td>Polymerase for Tag-Seq PCR</td></tr><tr><td>Primers (see text)</td><td>IDT</td><td>custom</td><td>PAGE purify Tag-Seq primers</td></tr></tbody></table>

massively parallel reporter assays in cultured mammalian cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

MPRAは、高解像度、転写調節エレメントの配列 - 活性関係の定量的な切開を容易にする。成功MPRA実験は、一般的にトランスフェクトライブラリ（ 図3A）中の配列の大部分を再現性の高い測定結果が得られます。乏しい再現性（ 図3B）が観察される場合、これは、アッセイされた配列のうち、1）低い絶対活性、または2）低いトランスフェクション効率のいずれかに、回収されたRNA試料中のレポーターmRNAの低すぎる濃度の指標である。 図4は 1,2センダイウイルスへの曝露の有無にかかわらず、HEK293細胞におけるヒトIFNB遺伝子の上流145 bpの配列の〜37000のランダムな変異型を分析することによって生成された代表「情報フットプリント」を示しています。プロモーターTATAボックスと知られている近位エンハンサー10は、明らかに情報豊富なレジとして識別することができるウイルス依存的にアドオン。 <img alt="図3" src="/files/ftp_upload/51719/51719fig3highres.jpg" /> 図3。タグ配列の再現性。高い（A）と低（B）再現性を持つ2つの独立した複製のトランスフェクションからのタグ配列データの例を示す散布図。後者のプロットは、2つの複製の一方のみに高いmRNAのカウントを持つ多くの外れ値のタグを表示します。このような成果物は、一般的に、レポーターmRNAの濃度によるレポーター構築物のうち、低い絶対活動、または低トランスフェクション効率のいずれか、定量的PCR増幅のために低すぎることを示している。 <img alt="図4" src="/files/ftp_upload/51719/51719fig4highres.jpg" /> 人間IFNB転写開始部位および近位エンハンサーの図4の情報フットプリント。 </strオング&gt;人間IFNB遺伝子の上流145ヌクレオチド（nt）の地域の約37000のランダムな変異体は、（A）とし、センダイウイルスに（B）の露出せずにHEK293細胞にMPRAを使用してアッセイした。青いバーは、レポーター出力し、各位置のヌクレオチド間の相互情報を表示します。近位エンハンサーおよびTATAボックスは、ウイルス感染時に高い情報コンテンツの領域として際立っている。

Watch this Scientific Journal Video about Massively Parallel Reporter Assays in Cultured Mammalian Cells at JoVE.com

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

培養哺乳動物細胞での超並列レポーターアッセイ

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

massively-parallel-reporter-assays-in-cultured-mammalian-cells

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JoVE Journal

Biology

21.5K ビュー.  Broad Institute. The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

ビデオ: Massively Parallel Reporter Assays in Cultured Mammalian Cells

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

マイクロスケール細胞刺激のための実験のための多目的自動化プラットフォーム

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

生物工学

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

光橋マッピングおよび生きている哺乳類細胞における Bioorthogonal 化学の Gpcr にプローブを化学物質の遺伝的結合の最適化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

培養細胞における癌関連シグナル伝達を問い合わせるための多重ルシフェラーゼベースのスクリーニング·プラットフォーム

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

高スループット/高含有スクリーニングに適応した細胞内マイコバクテリアの定量化のための顕微鏡表現型アッセイ

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

免疫・感染学

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

哺乳類細胞におけるイントロニックマイクロRNA成熟を解析するためのレポーターアッセイ

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

がん研究

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高スループット、リアルタイム、細胞内抗菌活性のデュアル読み出しテストおよび真核細胞の細胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

蛍光顕微鏡法による、複数世代にわたってライブ哺乳類細胞におけるプラスミドの複製を監視

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

生物学

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 ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

工学的HEK細胞を用いたMRGPRX2を活性化するペプチドのスクリーニング

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

細胞毒性の継続とリアルタイム監視のための自律的に生物発光哺乳類細胞

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...