The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

EMT誘導の1。細胞培養注：EMTは、上皮細胞におけるTGFβの治療または転写因子ツイストの異所性発現、カタツムリ、またはZEB1 / 2によって誘導することができる。このプロトコルで9,11,29不死化ヒト乳房上皮細胞（HMLE /ツイスト-ER、博士Jヤン、UCSDの贈り物）でのツイスト-ER融合タンパク質の発現を介して誘導性EMTシステムについて説明する。 4 -ヒドロキシタモキシフェン（TAM）処理すると、融合タンパク質ツイスト-ERは12〜14日9,11,29フルEMT遷移に転写し、その結果を駆動するために核に移行。そのようなHMLE /カタツムリ-ER、HMLE /TGFβ、およびMCF10A /TGFβのような追加のEMT誘導系は、これまでの出版物11,30に記載されています。 <ol><li> 5％のCO 2で37℃のインキュベーター内で無血清乳房上皮細胞成長培地（MEGM）にHMLE /ツイスト-ER細胞を維持します。継代の細胞を2〜3日毎にそれらを80％コンフルエンスに達したとき。 5〜10分間、37℃で0.15％トリプシンで細胞をインキュベートし、次いで、5％ウシ血清/ DMEMでトリプシンを不活性化する。細胞をスピンダウンし、MEGMで再懸濁します。 1〜4個の割合で新しい組織培養皿で細胞をプレート。 </li><li> 200μMの溶液を作製するためにエタノールにTAMに溶解する。 -20℃で、光とストアからTAMを保護します。 20 nMのTAMの最終濃度を作るために10,000希釈：使用前に、たて1でMEGM培地にTAMを追加します。 </li><li> EMTの誘導のために、重複して10cmの組織培養皿に1.6×10 6 HMLE /ツイスト-ER細胞をプレート。 20 nMのTAM含有MEGM培地で培養細胞。 </li><li>重複して10cmディッシュにプレート1.6×10 6 HMLE /ツイスト-ER細胞と非TAM処理の対照として0.01％エタノールで細胞を処理。 </li><li>二日間のインキュベーション後に、細胞の形態学的変化を記録するために10倍の倍率で光学顕微鏡下で写真を撮る。 </li><li>制御の一皿から培地を吸引したりTAM処理された細胞。 RNA単離およびタンパク質分析のためのRIPAバッファー中で、残りの半分のためのRNA溶解緩衝液中にプレートの半分を廃棄することにより、冷PBSで細胞を、収穫細胞を洗浄する。 </li><li>重複して10cmディッシュにおける再メッキ1.6×10 6細胞による制御またはTAM処理した皿から継代細胞。連続して20 nMのTAMや車両で細胞を処理する。 </li><li>繰り返しますビヒクル処置対照細胞が敷石状の上皮形態を維持する一方で、その時点で、TAM処理されたツイスト-ER細胞は、紡錘状の間葉の形態を示し、14日目まで5回隔日〜7を繰り返します。 </li></ol> EMTの2キャラ注：EMTの完了は次式で示されます。紡錘状の線維芽細胞のような外観に石畳状の上皮形態から（1）細胞の形態学的変化; （2）細胞間結合におけるE-カドヘリンの局在の喪失;損失によって定義されたEMTマーカーの（3）切り替え式上皮マーカーおよび間葉マーカーのゲイン。 細胞形態学的変化がEMT誘導の時間経過の間に10倍の倍率で光学顕微鏡によって捕捉され、細胞接合部におけるE-カドヘリンの節で説明1免疫蛍光検出は、このセクションに記載されている。 EMTマーカーの発現を免疫ブロット法によって検出される。一般的な上皮マーカーはE-カドヘリン、γ-カテニン、およびオクルディンであり、間葉マーカーは、フィブロネクチン、N-カドヘリンおよびビメンチンを含む。イムノブロット法の一般的な手順は、セクション4で説明されている。 <ol><li> TAM処理、種子を24ウェルプレートのウェルに入れ12mmのガラスの円形カバースリップ上で1×10 5細胞の12日目に。 </li><li> 48播種後時間、吸引媒体及び予め温めPBSで細胞を洗浄する。 </li><li> 15分間予め温め、4％パラホルムアルデヒドで細胞を固定します。 </li><li> PBSで細胞を3回洗浄する。 </li><li> 15のためにPBS中0.2％トリトンX-100で細胞をインキュベートRTで分。 </li><li> PBSで細胞を3回洗浄する。 </li><li>非特異的結合をブロックするために室温で1時間、5％BSA / PBSで細胞をインキュベートする。 </li><li>加湿チャンバーOの中で/ N 4℃で1％のBSA / PBSで1:50に希釈した一次抗体、E-カドヘリンと細胞をインキュベートします。注：一次抗体の希釈の範囲は1時50分〜1の間、通常は次のとおりです。200、および各抗体のための最適な希釈は、実験的に決定されるべきである。 </li><li>一次抗体をデカントし、PBSで細胞を3回洗浄する。 </li><li> RTで1時間500希釈：1中の蛍光標識二次抗体で細胞を培養する。この段階からは、光から細胞を保護する。 </li><li>二次抗体をデカントし、PBSで3回洗浄する。 </li><li> 10分間の0.1〜1グラム/ mlのDAPIまたはヘキストで染色細胞。 </li><li> PBSで細胞を3回洗浄します。 </li><li>マウントはマニキュアと封入剤の液滴と、シールカバースリップでカバースリップ。 </li><li>細胞を観察し、取る63X蛍光顕微鏡画像。 </li></ol>付加的な抗体を用いた染色は、EMT表現型を確認するために行うことができる。例えば、N-カドヘリンおよびα-平滑筋アクチンは、間葉表現型29,31,32のために染色することができる。細胞骨格構造の再編成は、F-アクチン11ファロイジンで細胞を染色することによってモニターすることができる。また、細胞運動性および細胞死抵抗性についてのアッセイは、EMT 11の特性を調べるために行うことができる。 定量RT-PCRアッセイを用いて検出スプライスアイ​​ソフォームの3。 <ol><li>プライマー設計 注：プライマー対は、慎重に異なるスプライスアイ​​ソフォームを増幅するように設計されるべきである。エクソンは、最も一般的に観察された選択的スプライシングイベントをスキップし、プライマー設計の戦略を説明するために例として示されている。 図1Aは、可変エクソンのような第三のエクソンと4つのエクソンプレmRNAを示している。エクソンの包含3結果エクソン3のスキッピングのに対し、より長いスプライスアイ​​ソフォームの生産は、短いスプライスアイ​​ソフォームを生成します。プライマーは、エクソン3に含まれるアイソフォームおよびエクソン3スキップさアイソフォームを区別するために、およびこのmRNAの全転写産物を検出するように設計される必要がある。 <ol><li>エクソン包含を検出するために、慎重に可変エクソン含まアイソフォームの特異的増幅を可能にする、可変エクソン3、近隣の構成的エクソン4内のリバースプライマー内部フォワードプライマーを設計します。フォワードの両方を設計し、ゲノムDNAまたはプレmRNAから増幅されたPCR産物を避けるために、同じ変数エクソン内のプライマーをリバースしないようにしてください。 </li><li>特にエクソンスキッピングイベント、変数エクソン3が含まれている場合には他の方法で破壊される構成的エクソン2及びエクソン4の接合部にまたがるプライマーの設計1を増幅した。このように、構成的エクソン4内の他のプライマーを設計し、PCR産物は、可変エクソン3がexcludである場合にのみ生成されますエドと構成的エクソン2及びエクソン4は、続いて接合されている。 </li><li>遺伝子の全転写物を検出するために、したがって、2つの構成のエクソン1及び2内のプライマーを設計し、PCR産物は、すべてのアイソフォームから増幅される。 </li><li>必ず全てのプライマーの融解温度（Tm）が約55°C、各プライマーの長さは約18〜22ヌクレオチドであることを確認し、PCR産物のサイズは80〜150塩基対である。 </li></ol></li><li> RNA抽出：全RNA単離キットを用いてRNAを抽出し（材料の表を参照）。 <ol><li> 350μlのRNA溶解バッファーで細胞を回収します。 </li><li>溶解物に70％エタノール350μl添加し、十分に混合します。 </li><li> RNA精製カラムにサンプルを転送します。 </li><li> 1分間のを10,000×gで遠心し、フロースルーを捨てる。 </li><li> RNA洗浄緩衝液IIで二度私は、RNAの洗浄緩衝液500μlで一回カラムを洗浄して。 </li><li>残留するRNA洗浄バッファーを削除する2分間最高速度で遠心分離することにより、カラムからII。 </li><li>新しい1.5 mlチューブにカラムを移し、カラムマトリックスの中央に50μlのヌクレアーゼフリー水を添加し、最高速度で遠心分離してRNAを溶出する。 </li></ol></li><li>第一鎖cDNAの合成<ol><li> UV吸収測定によりRNAの濃度と品質を決定します。注：良質のRNAは、約2.0の260nmで/ 280 nmの吸光度比を持つ必要があります。 </li><li> 20μlの最終容量中250 ngの全RNA、0.05μgのランダムヘキサマープライマー、50pmolのMgCl 2を 、10ピコモルのdNTP、2μLの5×GoScriptバッファー、及び1μlのRT酵素が含まれている逆転写酵素（RT）反応を設定します。 </li><li> RT酵素を不活性化するために5分間70℃でインキュベートし、1時間、42℃でRT反応を行います。 </li></ol></li><li>リアルタイムPCR <ol><li> 10μlのSYBRグリーンマスターミックスで構成されて20μlの反応を設定し、0.1〜1.0μlのcDNAテンプレート、および5フォワードピコモルおよびリバースプライマー。 </li><li>以下のステップを40サイクルでリアルタイムPCRを実行します。95℃の変性を10秒、10秒、58℃のアニーリング、および30秒間60℃の拡張のために。三重でPCR反応を実行します。 </li><li>解離曲線を確認し、単一のピークが各プライマーセットについて観察されることを確認してください。 </li><li>増幅曲線の二次導関数値を計算することによって定量化サイクル（CQ）値を得る。 </li><li> 「デルタデルタはCq（ΔΔCq） &#39;は、次式に示すような方法を使用して、非誘導サンプルと比較して、誘導されたサンプル中の標的mRNAの相対量（RQ）を算出する。参照として、そのようなGAPDHまたはTBPのようなハウスキーピング遺伝子を使用します。 </li></ol></li></ol><img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> より正確にスプライスアイ​​ソフォームの相対値を算出し、tの彼は考慮されるべき複数の参照遺伝子の使用。結果は、Pfaffl ら 33,34により記載された修飾絶対値の定量法を用いて分析することができる。 タンパク質レベルでのスプライスアイ​​ソフォームの4。審査<ol><li> 100μlの氷冷したRIPA緩衝液中で細胞を回収します。 </li><li>約10分間、氷上で溶解物を設定してください。 </li><li> 4°Cで10分間14,000×gで遠心分離し、上清を収集する。 </li><li>ブラッドフォードアッセイによってタンパク質濃度を測定する。 </li><li> SDSローディング緩衝液中のタンパク質試料を希釈し、10％SDS-PAGEゲル中のタンパク質の20〜40μgのを読み込む。 </li><li> 1.5時間20mAでSDS-PAGEゲルを実行します。 </li><li> 85 V.で3時間、4℃での湿式転送システムでPVDF膜へトランスファータンパク質は、標的タンパク質の分子量に応じて転送時間を調整する。 </li><li> RTで1時間30分間、5％無脂肪乳でブロック膜。 </li><li>メンブレンWIインキュベート4℃のCO / Nにおける一次抗体番目。 200〜1：5000、実験的に各抗体のための最適希釈を決定する一次抗体の希釈の範囲は、通常1との間である。このプロトコルで使用される抗体の希釈は、「特定の試薬や機器一覧表」に記載されています。 <ol><li>スプライスアイ​​ソフォームを検出するために、特定のアイソフォームを認識する特異的抗体を使用しています。別の方法として、構成的エクソンコード領域中のエピトープを認識する抗体を使用し、それらの異なるタンパク質の大きさに基づいて、同時にスプライスアイ​​ソフォームを検出します。 </li><li>同時に、EMTマーカーについて免疫ブロッティングによりEMTを監視します。間葉系マーカーとして使用するE-カドヘリン、γ-カテニン、および上皮マーカーとしてオクルディン、およびフィブロネクチン、N-カドヘリンおよびビメンチン。 </li></ol></li><li> 5分間隔でTBS-T（50mMのトリス、150mMのNaCl、0.05％ツイーン20、pH7.4）で洗浄膜は三回。 </li><li> HRP結合二次蟻を有する膜をインキュベートRTで1時間10,000希釈：1 ibody。 </li><li> 5分間隔でTBS-Tで洗浄膜は三回。 </li><li>化学発光検出システムを用いてタンパク質を可視化し、オートラジオグラフィーフィルムに膜を公開します。 </li></ol>

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</ol>

The authors declare that they have no competing financial interests.

ここで説明する手順は、誘導性EMTモデルにおける選択的スプライシングの検出を可能にする。このように、スプライスアイ​​ソフォーム発現の動的変化は、EMTの時間経過を通して捕捉することができる。非関連の細胞株とは異なる遺伝的背景が過度選択的スプライシングに影響を与える可能性があるため、この方法は、選択的スプライシングの比較のために異なるepithelial-または間葉表現?...

上皮間葉転換（EMT）は、胚発生の間に臓器の形態形成や組織リモデリングを駆動発生プログラムです。異常に活性化されると、EMTは腫瘍転移および臓器線維症1,2を推進しています。説得力のある研究はcobble-が失われ、接着結合タンパク質E-カドヘリンの発現を抑制するなどのツイスト、カタツムリ、およびZEBなどのいくつかの転写因子、によって定義されたEMT過程において転写調節の重要性を記載している上皮形態と紡錘形の間葉表現型3-8のゲインのような石。 RNAのゲノムワイド解析による最近の研究では、スプライシングパターン上皮または間葉表現型9,10のいずれかと関連している遺伝子群が存在することを明らかにした。私たちの研究室からの作業は、機能的に選択的スプライシングとEMTを接続。細胞表面接着分子CD44を研究することによって、私たちはCD44 alternことが実証されativeスプライシングは、しっかりとEMTの間に規制され、より重要なのは、因果的に切り替えることがCD44スプライスアイソフォームは11 EMTに貢献します。 ヒト多エキソン遺伝子の95％までは、あるいは12〜14にスプライシングされる選択的スプライシングは、遺伝子調節の普及と保存されたモデルを表す。単一の遺伝子から複数のタンパク質産物を生成することにより、選択的スプライシングは、ヒトゲノムの複雑さの別の層を追加すること、タンパク質の多様性のために必須の機構を構成する。このように、選択的スプライシングの調節不全は、潜在的にヒトの疾患の原因となる重大な生物学的効果につながる可能性があります。実際、疾患における異常な選択的スプライシングはスプライソソーム機構をコードする遺伝子の変異は、一般的に骨髄異形成症候群26-28に見られる最近の知見を含め、十年以上15-25文書化されています。従って、選択的にスプライシングされたiを検出するための信頼できる方法を開発するsoforms EMTを含む多様な生物学的プロセスの研究に非常に重要である。 ここでは、誘導性EMTモデルを用いて選択的スプライシングの変化を検出するためのプロトコルを提供する。 PCRプライマーを設計し、スプライスアイ​​ソフォームを検出するための方法は、EMTの間の選択的スプライシングの研究のためだけでなく、他の生物学的プロセスにおける選択的スプライシングの研究のためのみならず、適当である。 EMTの間に​​選択的スプライシングを調査することは、より良い、癌の転移を治療するための効果的な戦略の開発を促進し、EMTおよび腫瘍転移のメカニズムを理解するために不可欠である。

<table><tbody><tr><td>4-hydroxytamoxifen</td><td>Sigma</td><td>H7904</td><td>Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 &amp;#956;M. Keep the solution at -20 &amp;#176;C protected from light.</td></tr><tr><td>E.Z.N.A. Total RNA Isolation kit</td><td>Omega Bio-Tek</td><td>R6731</td><td>Total RNA isolation kit</td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5001</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>LightCycler 480 Real-Time PCR System</td><td>Roche</td><td></td><td>Equipment for qRT-PCR assay</td></tr><tr><td>CD44 antibody</td><td>R&amp;amp;D Systems</td><td>BBA10</td><td>1:1,000 dilution</td></tr><tr><td>E-cadherin antibody</td><td>Cell Signaling Technology</td><td>4065</td><td>1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td></tr><tr><td>&amp;gamma;-Catenin antibody</td><td>Cell Signaling Technology</td><td>2309</td><td>1:1,000 dilution</td></tr><tr><td>Occludin antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-5562</td><td>1:500 dilution</td></tr><tr><td>Fibronectin antibody</td><td>BD Transduction Laboratories</td><td>610077</td><td>1:5,000 dilution</td></tr><tr><td>N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>610920</td><td>1:2,000 dilution</td></tr><tr><td>Vimentin antibody</td><td>NeoMarkers</td><td>MS-129-p1</td><td>1:500 dilution</td></tr><tr><td>GAPDH antibody</td><td>Millipore Corporation</td><td>MAB374</td><td>1:10,000 dilution</td></tr><tr><td>Amasham ECL Western blotting detection reagent</td><td>GE Health Life Science</td><td>RPN2209</td><td>Chemiluminescence system</td></tr></tbody></table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

上記の手順は、EMTの間に​​選択的スプライシングを検出するための堅牢な方法を提供する。ツイストに誘導されるEMT時のCD44スプライスアイ​​ソフォームの切り替えの代表的な結果は、一例として以下に示す。 HMLE /ツイスト-ER細胞でのツイストによって誘導されるEMTは、細長い線維芽細胞表現型への上皮表現のような石畳、石からの移行（ 図2A）、上皮?...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

上皮間葉転移の間の選択的スプライシングの検出

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.9K ビュー.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

ビデオ: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

に薬剤耐性に関連する新規スプライス変異体を検出するために、RNA配列決定を使用して、<em&gt;インビトロ</em&gt;がんモデル

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

がん研究

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

具体的にはヒト細胞における選択的スプライシングを操作するために工学人工要因

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

遺伝学

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

代替の Pre-mrna スプライシング RNA In Situハイブリダイゼーション法によるマウス脳のセクションでの定量的解析

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

肉腫細胞における間葉上皮移行の誘導

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

発生生物学

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

乳がんおよび正常細胞におけるTGF-βシグナル伝達とTGF β誘発性上皮間葉間質転移の研究

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

E1Aミニジーンツールを使用したmRNAスプライシング変更の研究

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

スプライシング効率を監視するためのレポーターベースの細胞アッセイ

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

生化学

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

生物学

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

RNA-seqデータにおける選択的スプライシングおよびポリアデニル化の同定(英語)

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

RNAスプライシング

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...