Name: silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells
Uploaded: 2015-08-12T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1.のsiRNA溶液を調製します<ol><li>再懸 ​​濁5スクランブルのナノモル（Ctrlキー）とRNaseフリー水（最終siRNA濃度：50μM）の100μl中BRCA2 siRNAを乾燥したペレットの5ナノモル。これは、siRNAの株式であり、10μlのアリコートで-20℃で保存する必要があります。 </li><li> siRNAのストックから10μLのアリコートを取り、10μMのsiRNA作業溶液を得るために、RNaseフリー水40μlを添加します。この希釈は、使用するまで-20℃で保存する必要があります。 注：siRNAの作業溶液を3回以上解凍/凍結受けるべきではありません。 </li></ol>ヒト上皮細胞株の2播種<ol><li> 37℃、5％CO 2の加湿インキュベーター中で組織培養プレート中の細胞単層として増殖するヒト上皮細胞株から増殖培地を取り除き。 </li><li> RT PBS（100ミリメートルプレートのための10mlのPBS）で1回プレートに細胞を洗浄します。 </li><li>プレートから細胞を剥離するためには、0.25％トリプシン/ 0.53 mMのEDTA溶液2mlを添加し、細胞型に応じて3~5分間インキュベートします。 </li><li>剥離した細胞を回収する前に、10％ウシ胎児血清を含む完全増殖培地2mlを添加することによりトリプシンを中和します。 </li><li> 15mlチューブに細胞を移します。 </li><li>スイングバケットローターで4℃で3分間、320〜330×gで細胞を遠心します。 </li><li>抗生物質を含まない培地5ml中に細胞ペレットを再懸濁します。 </li><li> Burkerチャンバー中または製造者の指示に従って、自動計数装置を用いて顕微鏡下で細胞を数えます。 </li><li> 6ウェルプレート中で細胞を24時間（0.5〜1×10ウェルそれぞれについて、培地2mlで6個の細胞）の後に約80％コンフルエントになるように種子。播種する細胞の最適数は、特定の細胞株および成長速度に応じて変化し得ます。 注：抗生物質が負transfeの効率を妨害するので、この時点では、培地中の抗生物質を追加しないでくださいction。これは、BRCA2のsiRNAトランスフェクションの高い効率を達成するために、低培養継代（&lt;10）の細胞であることが非常に重要です。 </li></ol> siRNAで3トランスフェクト細胞<ol><li>二十四時間細胞をプレーティングした後、トランスフェクションの最初のラウンドに進みます。 <ol><li>低血清培地130μlの脂質ベースの10特異的なsiRNAトランスフェクション試薬の6μLを希釈します。 </li><li>低血清培地130μlの10μMのsiRNA（30ピコモル）の3μLを希釈します。 </li><li>希釈されたトランスフェクション試薬で希釈したsiRNAを組み合わせて、室温で5〜10分間インキュベートします。 </li><li>インキュベーションの間、細胞から培地を除去し、37°C​​（抗生物質を含まない）に予め温めておいた新鮮な培地2mlを加えます。 </li><li> （この量は6ウェルプレートの1ウェルのためのものである）を細胞にsiRNAをトランスフェクション試薬複合体の250μlを添加します。 </li><li> 37の加湿インキュベーター中で細胞をインキュベート76; C、5％CO 2。 </li></ol></li><li> 24時間後、ステップ3.1.2には、次の変更で3.1.1-3.1.5の手順を繰り返すことにより、トランスフェクションの第二ラウンドを続行：低血清培地130μlの10μMのsiRNA（50ピコモル）の5μLを希釈します。 <ol><li>分析の前に、37℃で1〜2日間細胞をインキュベートします。 注：トランスフェクションし、第二ラウンドで高いのsiRNA /トランスフェクション試薬比の使用の2ラウンドは、BRCA2サイレンシングの高効率を得るために不可欠です。 </li></ol></li></ol>イムノブロット分析のための4を溶解細胞<ol><li>非イオン性、非変性洗剤オクチルフェノキシポリエトキシエタノール、5mMのピロリン酸ナトリウム、1mMのオルトバナジン酸ナトリウム、10mMのフッ化ナトリウム、2mMのフェニルメチルスルホニルフルオリド、を10μg/ mlのロイペプチン、10を1％含有PBS新鮮な溶解緩衝液（pH 7.4）を準備/ mlのアプロチニン。氷の上に保管してください。 </li><li>細胞およびWからメディアを削除しますプレート内の冷PBS（2ミリリットル/ウェル）で1回、それらを灰。任意のPBSのキャリーオーバーを防止するために、完全にプレートから取り外します。 </li><li>各ウェルに200μlの溶解緩衝液を追加します。 注：細胞は、他のアッセイにも使用する必要がある場合、ステップ2.1〜2.6に記載し、使用する用途に応じて適切な緩衝液/培地中で細胞を再懸濁したように、細胞をトリプシン処理。 </li><li>静かに均一に溶解バッファーを配布し、回転子に4℃で15分間インキュベートする板を回転させます。 </li><li>セルスクレーパーを用いて氷上でマイクロ遠心チューブ内の細胞溶解物を収集します。 </li><li> 4℃で25分間、17,900×gで遠心分離し、新しいチューブに上清を収集します。 </li><li>製造業者の指示に従って市販のタンパク質アッセイを用いてタンパク質濃度を測定します。 </li></ol> 5. BRCA2のイムノブロット分析<ol><li> Bを実行して20倍のトリス - 酢酸SDSの50ミリリットルを希釈することにより、ランニング緩衝液を調製します蒸留水950ミリリットルでuffer（50mMのトリシン、50mMのトリス塩基、0.1％SDS、pHは8.24）。 </li><li>次のように試料を調製：20の最終容量まで15全細胞溶解物のμgの、4×サンプルバッファー5μlのpHは8.4でドデシル硫酸リチウムを含む、10倍のサンプル還元剤（0.5 M DTT）を2μl、および溶解バッファーを追加μlの。 注：（5μgのまで）全タンパク質のより少量を使用した場合、このプロトコルは、正常細胞株でBRCA2の検出を可能にします。 </li><li> 10分間55℃でサンプルを変性。 注：これは、55℃を使用することが重要です。 BRCA2タンパク質は感熱11であるため、より高い変性温度は、無傷の完全長（390キロダルトン）BRCA2タンパク質の一貫した損失をもたらします。 </li><li>製造者の指示に従って電気泳動チャンバーを設定します。 </li><li>サンプルおよびプレキャストゲル（トリス - 酢酸3から8パーセント）の高分子量プレ染色タンパク質標準の10μLをロードし、AB、120 Vで動作します2時間12アウト。 55kDaの青いマーカーがプレキャストゲルを離れる前に、電気泳動実行を停止します。 </li><li> 20X転送バッファの50ミリリットル、100％メタノール850 mlの水100mlで転送バッファを準備します。 </li><li>蒸留水で洗浄し、少なくとも5分間、転写緩衝液中で平衡化し、5分間、100％メタノール中に浸漬することによりPVDF膜（0.45μmの孔径）をアクティブにします。使用するまで4℃で転送バッファ内の転送装置のスポンジを浸します。 </li><li>電気泳動の実行が終了すると、次の順序（ボトムアップ）での転送スタックのサンドイッチ組み立てる：転送バッファに飽和3スポンジを、1 3MMグレード紙は、転送バッファ、ゲル、活性化されたPVDF膜、1 3mmの飽和しますトランスファー緩衝液に飽和等級紙、転送バッファ13,14飽和3スポンジ。装置内にスタックを配置し、4℃で一晩4℃で180 mAで4時間、350 mAでタンパク質を転送します。 注：ビーイングBRCA2非常に大きなタンパク質は、高分子量のタンパク質の完全な移動を確実にするために、4時間または一晩に1時間（メーカー、最も転送プロトコルによって示唆されるように）からの転送時間を延長することが重要です。また、原因長い転送時間には、過熱を最小化し、サンドイッチ分解を容易にするために、4℃の冷室でブロッティングを行うことが必須です。セミドライトランスファーシステムは、大きなタンパク質を転送するための良いものではありません。 </li><li>転写後、TBSで膜を洗浄し、0.25％（TBS-T）を30μlの抗BRCA2ウサギポリクローナル抗体と共に4℃で一晩、5％脱脂粉乳およびプローブを含むTBS-Tween中で室温で1時間、それを阻止します（ [：300（v / v）の希釈液1] 9ミリリットルTBS-T + 5％脱脂粉乳中で希釈した200μg/ ml）を、。 </li><li>その後TBS 10mlに希釈した西洋ワサビペルオキシダーゼ結合抗ウサギ二次抗体の1μlの1時間、フィルターをインキュベートし、TBS-Tを用いて膜を3回（10分毎）で洗浄-T + 5％の脱脂粉乳。その後、TBS-Tで膜を3回（各10分）を洗浄します。 </li><li>製造業者の説明書に従って、増強化学発光（ECL）ウエスタンブロッティング検出試薬を用いて、BRCA2タンパク質を明らかにし、ECLによって免疫検出を実行します。高解像度ECLフィルム上immunochemiluminescentバンドを可視化。 </li><li> 10 1,000抗チューブリン抗体（10μL：1に希釈したチューブリンをβに対するモノクローナル抗体を用いて、ローディングコントロールとして同じフィルタ上に、βチューブリン（55kDaの）のように、ハウスキーピング遺伝子に対する抗体を用いて免疫検出を行いますRTで1時間mlのTBS-T + 5％脱脂粉乳）。西洋ワサビペルオキシダーゼ結合抗マウスの代わりに抗ウサギ二次抗体を使用したことを除き、洗浄し、BRCA2（5.10から5.11）について記載したように二次抗体をインキュベートします。 </li><li> immunochemiluminescentバンドと感動フィルムのデジタル画像を取得し、専用の画像解析ソフトウェアによって、バンドの強度を分析する（ 例えば。、ImageJの）。 </li></ol>

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The authors have nothing to disclose.

女性と男性の乳癌、卵巣癌、前立腺癌、膵臓癌、及びメラノーマ3,4を含むいくつかの癌型のリスク増加にBRCA2遺伝子リードの生殖細胞変異は、多くの研究は、生物学的機能を理解するために行われているのでBRCA2タンパク質の。これらの研究のほとんどが原因BRCA2のような巨大なタンパク質の分析における技術的困難に遺伝ベースの主です。 BRCA2タンパク質の発現をサイレンシ?...

BRCA2（乳癌感受性遺伝子-2）遺伝子は、リコンビナーゼ酵素のRad51 1の機能を調節することにより、DNA二本鎖切断の修復に重要な役割を演じる腫瘍抑制タンパク質をコードします。 BRCA2はまた、転写の調節および細胞周期の制御2に関係しています。 BRCA2遺伝子に生殖細胞変異は、男性に女性と前立腺癌における乳癌および卵巣癌に常染色体優性の感受性を誘導するだけでなく、他の癌タイプ3,4の素因。しかし、抑制またはDNA損傷5-7を引き起こす化学療法剤に対する癌細胞の脆弱レンダリングしてもよいBRCA2の機能を低下させる癌、BRCA2変異を開発するリスクの増加にもかかわらず。 BRCA2タンパク質レベルの低下は、そのBRCA2タンパク質を示唆し、いくつかの癌の種類で検出されているものの、散発性の癌はBRCA2変異 （&lt;3％）の低速度を示します非突然変異依存機構7,8を介して散発性の癌において腫瘍形成の間に失われることがあります。したがって、完全に癌生物学の文脈において、ならびに他の生物学的環境においてBRCA2の機能を理解することが重要です。 哺乳動物細胞における遺伝子の新規な機能を識別するために使用される強力なツールは、その発現を抑制することです。一例として、正常上皮細胞の様々なBRCA2の発現を抑制することは、最近アノイキス耐性の調節、癌細胞浸潤性および転移能9の取得中に重要なステップとして、BRCA2の新規機能の同定につながりました。遺伝子サイレンシングは、細胞小干渉（SI）RNAまたは特定の遺伝子を標的とする短いヘアピン（SH）RNA分子のいずれかで導入することによって達成することができます。高いsiRNAの効力とその使いやすさは重要な生物学的プロセスに新たな洞察を得ることを目的とした実験をサイレンシングするため、それを優先ツールを作ります新たな治療標的を同定するためにND。しかし、遺伝子発現の効率的なノックダウンは、すべての遺伝子のために容易に達成できないことがあり、細胞の種類に応じて非常に変化してもよいです。細胞内タンパク質のレベルの減少は、調査中の最も関連性の表現型であるため、遺伝子産物の解析をイムノブロッティングにより遺伝子サイレンシングを定量化することが重要です。これに関しては、BRCA2タンパク質は、さらに課題を提示：大きなタンパク質（約390キロダルトン）をされ、技術的な困難が免疫ブロット法を含む、従来の生化学的解析のために存在します。 ここでは、ヒト上皮細胞株におけるBRCA2の効率的なサイレンシングのためと分析をイムノブロッティングによりノックダウンBRCA2タンパク質の迅速かつ成功した検出のために最適化されたプロトコルを報告しています。

<table><tbody><tr><td>BRCA2 siRNA</td><td>Dharmacon</td><td>L-003462-00</td><td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td></tr><tr><td>Ctrl siRNA</td><td>Dharmacon</td><td>&amp;nbsp;D-001810-10-05</td><td>ON-TARGETplus Non-targeting Pool</td></tr><tr><td>Lipofectamine RNAiMAX Reagent&amp;nbsp;</td><td>Life Technologies</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/13778075&quot;&gt;13778075&lt;/a&gt;</td><td>Transfection Reagent</td></tr><tr><td>OPTI-MEM I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-070</td><td>Medium for transfection procedure</td></tr><tr><td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td><td>Life Technologies</td><td>EA0378</td><td>Gel for protein electrophoresis</td></tr><tr><td>NuPAGE LDS Sample buffer&amp;nbsp;</td><td>Life Technologies</td><td>NP0007</td><td>Reagent for protein electrophoresis</td></tr><tr><td>NuPAGE Sample Reducing agent (10x)</td><td>Life Technologies</td><td>NP0004</td><td>Reagent for protein electrophoresis</td></tr><tr><td>NuPAGE Antioxidant</td><td>Life Technologies</td><td>NP0005</td><td>Reagent for protein electrophoresis</td></tr><tr><td>HiMark Pre-stained protein standard</td><td>Life Technologies</td><td>LC5699</td><td>Prestained marker for gel electrophoresis</td></tr><tr><td>XCell SureLoc Mini-Cell&amp;nbsp; System</td><td>Life Technologies</td><td>EI0002</td><td>Equipment for protein electrophoresis/transfer</td></tr><tr><td>NuPAGE Tris-Acetate SDS Running Buffer</td><td>Life Technologies</td><td>LA0041</td><td>Reagent for Western blotting</td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td>Reagent for Western blotting</td></tr><tr><td>NuPAGE Transfer Buffer</td><td>Life Technologies</td><td>NP0006-1</td><td>Reagent for Western blotting</td></tr><tr><td>BRCA2 H-300 rabbit polyclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-8326</td><td>1:300 dilution</td></tr><tr><td>Beta-tubulin monoclonal antibody</td><td>Sigma Aldrich</td><td>T4026-100UL</td><td>1:1,000 dilution</td></tr><tr><td>Skim Milk powder</td><td>Sigma Aldrich</td><td>70166</td><td>Reagent for Western blotting</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379</td><td>Reagent for Western blotting</td></tr><tr><td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td><td>Pierce Thermo Scientific</td><td>34080/34096</td><td>Chemiluminescence system</td></tr><tr><td>PNT1A cells</td><td>SIGMA Aldrich (for ECACC)</td><td>95012614</td><td>PNT1A human, normal prostate epithelium immortalized with SV40</td></tr><tr><td>Nthy cells</td><td>SIGMA Aldrich (for ECACC)</td><td>90011609</td><td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td></tr><tr><td>Cell Proliferation Kit I (MTT)</td><td>Roche</td><td>11465007001</td><td>Kit for cell proliferation assays</td></tr></tbody></table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

従来の生化学的分析による腫瘍抑制タンパク質BRCA2とその検出のサイレンシングは、ヒトのBRCA2タンパク質（約390キロダルトン）のサイズが大きいために大きな技術的課題を表します。我々は、ヒト上皮細胞株において、それぞれ、ヒトのBRCA2を沈黙し、内因性BRCA2タンパク質を検出するための標準的なsiRNAトランスフェクションおよびイムノブロッティングのプロトコルの変更を報告しています。重要なステップは、トランスフェクション試薬の比が高いsiRNAを、同じ実験内のsiRNAトランスフェクションの2その後のラウンドがあります。抗BRCA2抗体を用いたイムノブロット分析によって判断されるように、我々は一貫して人間のBRCA2遺伝子の70％以上のサイレンシングを達成するための標準プロトコルへのこれらおよびその他の変更を使用しました。また、代わりに、従来の70〜100°Cおよび免疫ブロット法の他の技術的な最適化の55℃での細胞溶解物の変性は、無傷のBRCA2タンパク質の検出が可能であってもWH出発物質が非常に少量エン入手可能であるか、またはBRCA2タンパク質の発現レベルが非常に低い場合。ヒト細胞におけるBRCA2の効率的なサイレンシングは、腫瘍中の病原性BRCA2変異によって影響を受ける可能性がある新たな分子経路および細胞機能を調べるために、腫瘍細胞を含む、無傷のBRCA2と細胞内でBRCA2の機能を破壊するための貴重な戦略を提供しています。効率的なサイレンシングおよびBRCA2のような他の「大規模な」タンパク質の分析のためにこのプロトコルの適応が容易に達成する必要があります。

細胞の機能にサイレンシングBRCA2の影響を調べるために、生化学的/生物学的ア ​​ッセイに進む前に、最初のステップは、BRCA2のsiRNA（ 図1Aと並んでスクランブルsiRNA（非標的とするsiRNA）側を使用してサイレンシングの特異性を確認することです）。これは、BRCA2サイレンシングの高い効率を得るために高いのsiRNA /トランスフェクション比でsiRNAトランスフェ...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

のサイレンシング<em&gt; BRCA2</em&gt;ヒト培養細胞における新規BRCA2-調節される生物学的機能を同定するために、

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K ビュー.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

ビデオ: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

癌細胞スフェロイド培養における潜在的に新規薬剤標的を同定するためのスクリーニング機能ゲノミクスを利用

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

がん研究

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

CRISPR媒介ベース・エディタを用いた BRCA1 バリアントの機能評価

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

医学

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

がん薬感の有効性を評価<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

生物学

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

イン・ビトロ 標的エピジェネティックサイレンシングのための遺伝子操作転写リプレッサーの選択

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

生物工学

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

の全配列のNGS解析のためのトランスポゼースベースの技術によるgDNAのエンリッチメント<em&gt; BRCA1</em&gt;<em&gt; BRCA2</em&gt;、およびDNA損傷修復に関与する遺伝子9

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

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癌細胞スフェロイド培養における潜在的に新規薬剤標的を同定するためのスクリーニング機能ゲノミクスを利用

CRISPR媒介ベース・エディタを用いた BRCA1 バリアントの機能評価

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

がん薬感の有効性を評価

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

イン・ビトロ標的エピジェネティックサイレンシングのための遺伝子操作転写リプレッサーの選択

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

の全配列のNGS解析のためのトランスポゼースベースの技術によるgDNAのエンリッチメント

免疫蛍光によるDNA修復タンパク質相互作用の可視化

のサイレンシング

要約

さらに動画を探す

この動画の章

癌細胞スフェロイド培養における潜在的に新規薬剤標的を同定するためのスクリーニング機能ゲノミクスを利用

CRISPR媒介ベース・エディタを用いた BRCA1 バリアントの機能評価

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

がん薬感の有効性を評価

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

イン・ビトロ 標的エピジェネティックサイレンシングのための遺伝子操作転写リプレッサーの選択

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

の全配列のNGS解析のためのトランスポゼースベースの技術によるgDNAのエンリッチメント

免疫蛍光によるDNA修復タンパク質相互作用の可視化

イン・ビトロ標的エピジェネティックサイレンシングのための遺伝子操作転写リプレッサーの選択