この作品は、バージニアコモンウェルス大学研究所理事長奨励プログラム（PRIP）144602から3（PX）で、国立衛生研究所（PX）からの助成金によってR01DE018138と部分的にサポートされていました。我々は博士に感謝します。レイ·チェン、Yuetanドウとゲノムワイド変異体の構築をサポートするため、王をXiaojing。我々はまた、DNAシークエンシングのためにバージニアコモンウェルス大学でDNAの中核施設に感謝します。

1。プライマー設計<ol><li>プライマーはS.に基づいて家のスクリプトで使用して設計されてい血統 SK36ゲノム配列。プライマー、F1/R1、F2/R2、F3/R3と、3組の標的遺伝子の1-kbの上流配列を増幅するために設計されており、APHA-3をコードする遺伝子、カナマイシン耐性（キロr）はタンパク質3と1それぞれの標的遺伝子の-kbの下流配列、（ 図1）。これらのプライマーのうち、F1およびR3は、プログラムのEMBOSSスイート（でePrimer3を使用して設計されて<a href="http://emboss.sourceforge.net/apps/cvs/emboss/apps/index.html" target="_blank">http://emboss.sourceforge.net/apps/cvs/emboss/apps/index.html</a>ターゲットの上流または下流隣接領域を増幅するために）遺伝子。遺伝子特異的プライマー、R1とF3は、標的遺伝子の5 &#39;および3&#39;配列に基づいて設計されています。 R1とF3プライマーはAPHA-3に相補的であり、それらの5 &#39;末端の25-bpのアダプター配列を含んでいる遺伝子。各プライマーの融解温度は、できるだけ近い60になるように設計されました°C、96ウェルフォーマットで、すべてのPCR反応のため均一なアニール温度の使用を可能にする。 </li><li> F1、R1、F3とR3プライマーは遺伝子の順序に基づいて、96ウェルプレート内で合成される。各プレートは、同じ遺伝子の順序におけるプライマーの1種類が入っています。マルチチャンネルピペットを用いてPCR増幅のための10μMの最終濃度になるように96ウェルワーキングプライマープレート内のプライマーを希釈します。 </li></ol> 2。ハイスループットPCR増幅および精製<ol><li>ターゲットSの1-kbの上流または下流を増幅する高スループットへ血統の遺伝子は、1640μlのddH 2 Oを、250μlの10xHighフィデリティPCRバッファー、10mMのdNTP混合液200μl、50mMのMgSO 4を 、100μlの100μlを含む15 mlコニカルチューブ内の氷の上でPCRカクテル混合物を組み立てる10 ng /μlにS.血統 SK36ゲノムDNAおよび10μlのプラチナTaq DNAポリメラーゼpolyme撃つ高忠実度やマルチチャンネルピペットを用いて、96ウェルPCRプレートに各ウェルに23μlの混合物を転送します。マルチチャンネルピペットを用いて、PCRプレートにプライマープレートを作業10μMから各F1とR1（またはF3およびR3）の1μLを。 PCRプレートを密封し、1.5分間、30秒、68℃で30秒間、1分間、及び94℃の30サイクル、54℃、94℃で増幅を行う。 </li><li> 48ウェルフィッティングマルチチャンネルピペットのロードとエチジウムブロマイドを含む1％アガロースゲルを準備します。 96ウェルプレートに5xDNAローディングバッファー1μlを4μlのPCR産物を混合し、10μlのマルチチャンネルピペットを用いてアガロースゲルにサンプルをロードします。 30分間135 Vで電気泳動を実行します。 UVPのマニュアルおよび解析システムでゲル上のバンドを調べます。 </li><li>製造業者の指示に従って遠心分離を用いPureLink 96 PCR精製キットでPCR産物を精製する。 DNAを溶出するために、bのウェルに40μlの滅菌蒸留H 2 Oを追加プレートをinding。 </li><li>ランダム光度計、分光光度計を用いてDNA濃度を調べるために板の上にいくつかの精製されたアンプリコンを選ぶ。 〜10 ng /μlに、プレート上のアンプリコンの濃度を調整します。 </li><li>プラスミドを含むキロRカセットは PCR鋳型としてEcoRIにて Iを用いて消化される。消化したプラスミドをQIAquick PCR精製キットにより精製されており、線状プラスミドDNAは最終DNA濃度が10 ng /μlに調整されます。 4、10μM、F2、1μlの1μlのSO 16.4μlのddH 2 Oを、2.5μlの10xHighフィデリティPCRバッファー、10mMのdNTP混合液2μl、50mMのMgの1μlを含むキロRカセットリコン用の25μlの混合物を組み立てる10μMのR2は、1μlの線状プラスミドDNAを、0.1μlのプラチナTaq DNAポリメラーゼハイファイ。 ℃で1分間、30秒、68のために30秒で1分、そして94℃の30サイクル、55℃、94℃でPCR増幅を行う。 1％アガロースゲル上で電気泳動によりPCR産物を調べます。うんちQIAクイックPCR精製キットで10個々のPCR精製からキロRカセットリコンのラバルク。 10 ng /μlにするアンプリコンの濃度を調整します。 </li><li>最後の直線換えPCRアンプリコンを取得するには、3つのPCRアンプリコンはウェルPCRテンプレートとして1 PCRプレートの各1μlを（ほぼ等しいモル量で）と結合されます。他のPCRコンポーネント4、10μMのF1を1μl、10μMR3の1μlのは、SO 14.4μlのddH 2 Oを、2.5μlの10xHighフィデリティPCRバッファー、10mMのdNTP混合液2μl、50mMのMgの1μlを含めて追加されます0.1μLプラチナTaq DNAポリメラーゼハイファイ。 PCR増幅は4分間3.5分、最後に68℃で30秒、68℃30秒55℃2分間、94℃で30サイクル、94℃で行われる。 </li><li>アガロースゲル上のPCR増幅産物を調べます。上記のように、PCR産物を精製し、定量化する。 -20℃で再結合されたPCRアンプリコンを凍結</li></ol> 3。 COMpetent細胞の調製<ol><li>トッドヒューイットブロスを作製し、室温まで冷却し、0.22μmのポリスチレンフィルタ4を用いて滅菌した後沸騰するまで加熱10NのNaOHを用いてpHを7.6に調整。 TH + HS媒体を補うために、15 mlコニカルチューブに11.7ミリリットルトッドヒューイットブロスに300μlの熱不活化ウマ血清（2.5％最終濃度）を追加します。アリコートのTH + HS培地2ml及びチューブ内に10ミリリットル。 </li><li>株式Sの5μlを接種血統 SK36は2ミリリットルのTH + HS培地中に-80℃で凍結し、37℃でしっかりとプレインキュベート10ミリリットル-TH + HSチューブ伴う℃をキャッピングで一晩培養した培養液をインキュベートする。 </li><li>一晩後、10ミリリットルのTH + HSに50μlの培養を転送し、3時間（0.07から0.08のOD 660に対応）37℃でチューブをインキュベート、直ちに形質転換のために使用します。 </li></ol> 4。細胞の形質転換と抗生物質選択<ol><li> 70 ngのSの2μlを加え血統 SK36コンペタンス刺激ペプチド（CSP）および37のブロック96ウェル、それらプリ暖かい上のエッペンドルフチュー​​ブに2μlの線形リコンビナントPCRアンプリコン（〜50 ngの）℃各試験管に3時間インキュベートしたSK36文化の330μlを。 1時間37℃でインキュベートする。制御などの滅菌ddH 2 OでDNAを交換してください。 </li><li>氷の上にブロックを配置し、500μg/ mlのカナマイシンをブレインハートインフュージョン（BHI）寒天プレート上でそれぞれの変換の100μlを広げた。微好気条件下で2日間37℃でプレートをインキュベートする。 </li></ol> 5。変異体の確認とストレージ<ol><li>それぞれの置換変異体は、ランダムに、2個のコロニーを拾う500μg/ mlのカナマイシンを含む5mlのBHI培地にコロニーを接種し、37℃で一晩microaerobically接種をインキュベート℃に-80℃で30％グリセロールで各文化を凍結保存°C </li><li> 、予想される遺伝子置換を含む変異体は、それぞれの変異体を使用してF1とR3にはコロニーPCRを行うかどうかを調べるために、96ウェルPCRプレートのプライマー。個々のコロニーから約1μlの一晩培養物を25μlのPCR-増幅反応におけるDNA鋳型として使用されます。 PCRは℃で3.5分、最後に68℃で4分間、30秒、68で30秒、55℃で5分、94、35サイクルの°Cのために94℃で行われる。 </li><li>正確にはダブルバンド変異体又は汚染PCRアンプリコンを識別するには、エチジウムブロマイド染色で&gt; 12センチ2％アガロースゲル上で4時間電気泳動によってPCRアンプリコンを調べます。このアガロースゲル電気泳動条件の下で、≥100 bpの差を持つ任意のアンプリコンが明確に特定された。キロr のカセットと、野生型遺伝子の増幅に起因するバンドが&lt;100 bpによって異なることが予想されている場合、内部のT1プライマーは、野生型遺伝子をPCRにより検出することができるかどうかを判断するために使用されます。 </li><li>さらに削除を確認するには、アンプリコンはPureLink 96 PCR精製キットで精製し、そのP1プライマーを用いて配列決定されるキロRカセットに結合する。配列決定によって確認のみ正しい変異を保持します。 </li></ol>

<ol>
	<li>Sassetti, C. M., Boyd, D. H., Rubin, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+required+for+mycobacterial+growth+defined+by+high+density+mutagenesis.">Genes required for mycobacterial growth defined by high density mutagenesis.</a> Mol. Microbiol. 48, 77-84 (2003).</li><li>Kobayashi, K., 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=Essential+Bacillus+subtilis+genes.">Essential Bacillus subtilis genes.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 4678-4683 (2003).</li><li>Trieu-Cuot, P., Courvalin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+the+Streptococcus+faecalis+plasmid+gene+encoding+the+3'5"-aminoglycoside+phosphotransferase+type+III.">Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III.</a> Gene. 23, 331-341 (1983).</li><li>Paik, S., 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=Identification+of+virulence+determinants+for+endocarditis+in+Streptococcus+sanguinis+by+signature-tagged+mutagenesis.">Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.</a> Infect. Immun. 73, 6064-6074 (2005).</li><li>Lukomski, S., 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=Nonpolar+inactivation+of+the+hypervariable+streptococcal+inhibitor+of+complement+gene+(sic)+in+serotype+M1+Streptococcus+pyogenes+significantly+decreases+mouse+mucosal+colonization.">Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization.</a> Infect. Immun. 68, 535-542 (2000).</li><li>Baba, 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=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+the+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> Mol. Syst. Biol. 2, (2006).</li><li>de Berardinis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complete+collection+of+single-gene+deletion+mutants+of+Acinetobacter+baylyi+ADP1.">A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.</a> Mol. Syst. Biol. 4, 174 (2008).</li><li>Rodriguez, A. M., 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=Physiological+and+molecular+characterization+of+genetic+competence+in+Streptococcus+sanguinis.">Physiological and molecular characterization of genetic competence in Streptococcus sanguinis.</a> Mol. Oral. Microbiol. 26, 99-116 (2011).</li><li>Perry, D., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Streptococcus+mutans+Infect.">Genetic transformation of Streptococcus mutans Infect.</a> Immun. 32, 1295-1297 (1981).</li><li>Pakula, R., Walczak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+of+competence+of+transformable+streptococci.">On the nature of competence of transformable streptococci.</a> J. Gen. Microbiol. 31, 125-133 (1963).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Xu, 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=Genome-wide+essential+gene+identification+in+Streptococcus+sanguinis.">Genome-wide essential gene identification in Streptococcus sanguinis.</a> Sci. Rep. 1, (2011).</li></ol>

特別な利害関係は宣言されません。

初期のプライマーが設計しながら、隣接する遺伝子の外因性の抗生物質遺伝子と1標的遺伝子の交換の可能性極性効果を最小限に抑えるためには、2つのステップがとられる。削除された遺伝子のほとんどは、プライマーR1とF3は前に終止コドンから30 bpに開始コドンに続く6 BPからコーディング領域を削除するために設計されています。最後の30塩基対は、隣接する下流遺伝子によって使用され?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;名前</strong</td&gt;<td<strong&gt;会社概要</strong</td&gt;<td<strong&gt;カタログ＃</strong</td&gt;<td<strong&gt;コメント（オプション）</strong</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーF2</td&gt;<td&gt;統合DNA技術</td&gt;<td</td&gt;<td&gt; TGACTAACTAGGAGGAATAAATGGCTAAAATGAGAATAT</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーR2</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; CATTATTCCCTCCAGGTACTAAAACAATTCATCCAGT</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーF2P</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GATAAACCCAGCGAACCATTTGA</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーP1</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GCTTATATACCTTAGCAGGAGACA</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーP2</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GTATGACATTGCCTTCTGCGTCC</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーの5 &#39;end_R1_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GCCATTTATTCCTCCTAGTTAGTCA</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーの5 &#39;end_F3_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; GTTTTAGTACCTGGAGGGAATAATG</td&gt;</tr&gt;<tr&gt;<td&gt;プライマーの5 &#39;end_R1p_seq</td&gt;<td&gt;同上</td&gt;<td</td&gt;<td&gt; CCTCAAATGGTTCGCTGGGTTTATC</td&gt;</tr&gt;<tr&gt;<td&gt; CSP</td&gt;<td&gt; Synprep</td&gt;<td</td&gt;<td&gt;シーケンス：NH2-DLRGVPNPWGWIFGR、-COOH</td&gt;</tr&gt;<tr&gt;<td&gt; DNAポリメラーゼ</td&gt;<td&gt;インビトロジェン</td&gt;<td&gt; 11304-102</td&gt;<td&gt;プラチナTaq DNAポリメラーゼハイフィデリティ</td&gt;</tr&gt;<tr&gt;<td&gt;をEcoRI</td&gt;<td&gt;ニューイングランドバイオラボ社</td&gt;<td&gt; R0101S</td&gt;<td&gt;制限酵素</td&gt;</tr&gt;<tr&gt;<td&gt;寒天</td&gt;<td&gt;アメリカン·バイオ分析</td&gt;<td&gt; AB01185</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;アガロース</td&gt;<td&gt;プロメガ</td&gt;<td&gt; V3125</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; BHI</td&gt;<td&gt; BD Biosciences社</td&gt;<td&gt; 237500</td&gt;<td&gt;ブレインハートインフュージョン</td&gt;</tr&gt;<tr&gt;<td&gt;馬血清</td&gt;<td&gt;フィッシャー·サイエンティフィック</td&gt;<td&gt; SH3007403</td&gt;<td&gt;馬血清</td&gt;</tr&gt;<tr&gt;<td&gt;キロ</td&gt;<td&gt;フィッシャー·サイエンティフィック</td&gt;<td&gt; BP906-5</td&gt;<td&gt;カナマイシン</td&gt;</tr&gt;<tr&gt;<td&gt; THブロス</td&gt;<td&gt; BD Biosciences社</td&gt;<td&gt; 249240</td&gt;<td&gt;トッドヒューイットブロス</td&gt;</tr&gt;<tr&gt;<td&gt; PureLink 96</td&gt;<td&gt;インビトロジェン</td&gt;<td&gt; K310096</td&gt;<td&gt; PureLink 96 PCR精製キット</td&gt;</tr&gt;<tr&gt;<td&gt; QIAクイック</td&gt;<td&gt;キアゲン</td&gt;<td&gt; 28106</td&gt;<td&gt; QIAクイックPCR精製キット</td&gt;</tr&gt;<tr&gt;<td&gt;フィルター</td&gt;<td&gt;コーニング</td&gt;<td&gt; 431117</td&gt;<td&gt;0.22μmのポリスチレンフィルター</td&gt;</tr&gt;<tr&gt;<td&gt; Anoxomatシステム</td&gt;<td&gt;マート微生物BV</td&gt;<td&gt; AnoxomatマークII</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;ベンチトップ遠心分離機</td&gt;<td&gt;サーモサイエンティフィック</td&gt;<td&gt; 75004377</td&gt;<td&gt;ソーバル伝説RTプラスマイクロローター</td&gt;</tr&gt;<tr&gt;<td&gt;ゲルドキュメンテーション</td&gt;<td&gt; UVP LLC</td&gt;<td&gt; BioDocイット210</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;インキュベーター</td&gt;<td&gt;フィッシャー·サイエンティフィック</td&gt;<td&gt; 11から690-625D</td&gt;<td&gt; Isotemp</td&gt;</tr&gt;<tr&gt;<td&gt; LabnetのジェルXL</td&gt;<td&gt; Labnet</td&gt;<td&gt; E0160</td&gt;<td&gt; LabnetのジェルXL</td&gt;</tr&gt;<tr&gt;<td&gt;マイクロ遠心機</td&gt;<td&gt;エッペンドルフ</td&gt;<td&gt; 22621408</td&gt;<td&gt;マイクロ遠心機5415 R</td&gt;</tr&gt;<tr&gt;<td&gt;マルチチャンネルピペット</td&gt;<td&gt;サーモScientfic</td&gt;<td&gt; 4661040、4661020</td&gt;<td&gt;フィンピペットF1]</td&gt;</tr&gt;<tr&gt;<td&gt;サーマルサイクラー</td&gt;<td&gt;アプライドバイオシステムズ</td&gt;<td&gt; N805-0200</td&gt;<td&gt;ジーンアンプPCRシステム9700</td&gt;</tr&gt;</tbody&gt;</table

genome-wide gene deletions in streptococcus sanguinis by high throughput pcr

トランスポゾン突然変異誘発及び単一遺伝子欠失は細菌1,2におけるゲノムワイドな遺伝子ノックアウトで適用2つの方法です。トランスポゾン突然変異誘発は、低コスト、時間の無駄であり、完成したゲノム情報を必要としませんが、この方法で2つの弱点がありますが：低下競争を有する変異体をカウンターで選択された混合された変異体ライブラリー内の異なる変異体（1）の可能性が;と、（2）部分的な遺伝子の不活性化の可能性はそれによって遺伝子が完全にトランスポゾンの挿入後に、その機能を失うことはありません。単一遺伝子欠失解析は、トランスポゾン突然変異誘発に付随する欠点を補償することができる。ゲノムワイドな単一遺伝子欠失の効率を改善するために、私たちはモデル生物として連鎖球菌血統を使ってゲノムワイドな単一遺伝子欠失のためのハイスループット技術を確立しようとします。各遺伝子の欠失は、Sで構築血統ゲノムは1を備えるように設計されています標的遺伝子の-kbの上流に、標的遺伝子のカナマイシン耐性タンパク質をコードAPHA-3遺伝子、および1-kbの下流。それぞれのプライマーF1/R1、F2/R2、F3/R3と、、の3つのセットを設計し、各欠失構築物のこれらの3つのコンポーネントのPCR増幅のための96ウェルプレートフォーマットで合成される。プライマーR1とF3はそれらの5 &#39;末端APHA-3遺伝子の領域に相補的な25塩基対の配列を含む。 APHA-3遺伝子の大規模PCR増幅はすべて単一遺伝子欠失構築物を作成するために一回行われます。 APHA-3遺伝子のプロモーターは、当初カナマイシンカセットの潜在的な極性の影響を最小限に抑えるために除外されます。遺伝子欠失構築物を作成するには、ハイスループットPCR増幅および精製は、96ウェルプレートフォーマットで行われます。各遺伝子欠失のための線形換えPCRアンプリコンは、高忠実度DNAポリメラーゼを用いて4つのPCR反応を介して構成されます。初期EXPONSの動増殖期トッドヒューイットブロス中で培養した血統は、不活化ウマ血清をPCR-換え構築物の形質転換のために能力を高めるために使用される2.5％を補足。この条件では、Sの最大20％ 血統細胞は DNAの約50 ngを使用して変換することができます。このアプローチに基づいて、単一遺伝子欠失を持つ2048の変異体は、最終的にSの2270の遺伝子から得られたSに他のORF内に完全に含まれる4つの遺伝子のORFを除く血統血統 SK36と218潜在的な必須遺伝子。遺伝子欠失構築物を作成するための技術は、高スループットであり、任意の形質転換可能な細菌のゲノムワイドな単一遺伝子欠失で使いやすいかもしれません。

後のPCRプライマーF1とR1を使用して増幅し、F3およびR3は、それぞれSの約1 kbの上流および下流血統遺伝子は、96ウェルフォーマットであった（ 図2A）で得られた。私たちのPCR条件と使用して設計されたプライマーの下では、特定の製品はSから増幅した各PCR反応における血統ゲノムDNA。この結果は、プライマーがSのターゲットに非常に特異的であっ...

Watch this Scientific Journal Video about Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR at JoVE.com

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

効率的なゲノムワイドな単一遺伝子突然変異法は使用して確立されている<em&gt;ストレプトコッカス血統</em&gt;モデル生物として。この方法は、高スループット換えPCRとの変換を介して実現しています。

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 ...

genome-wide-gene-deletions-streptococcus-sanguinis-high-throughput

Research

JoVE Journal

Biology

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

16.4K ビュー.  Virginia Commonwealth University. 効率的なゲノムワイドな単一遺伝子突然変異法は使用して確立されているストレプトコッカス血統モデル生物として。この方法は、高スループット換えPCRとの変換を介して実現しています。

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

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 ...

生物学

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

ミッションターゲットIDのライブラリを使用してmiRNAのターゲットのゲノムワイドスクリーン

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

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. ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

複数の遺伝子欠失を持つ酵母菌株の世代のためのグリーンモンスタープロセス

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

生細胞内のタンパク質断片相補アッセイ（PCA）によるゲノムワイドなタンパク質間相互作用のスクリーニング

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

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 ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

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).

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）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-配列：アデニンメチル化DNA断片のハイスループットシークエンシングによるタンパク質-DNA相互作用のゲノムワイドなマッピング

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...