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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V., Timchenko, L. T., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+splicing+regulated+by+a+CUG-binding+protein+in+myotonic+dystrophy.">Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy.</a> Science. 280, 737-741 (1998).</li><li>Savkur, R. S., Philips, A. V., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+regulation+of+insulin+receptor+alternative+splicing+is+associated+with+insulin+resistance+in+myotonic+dystrophy.">Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy.</a> Nat Genet. 29, 40-47 (2001).</li><li>Spillantini, M. 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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 border="0" cellpadding="0" cellspacing="0" width="1167">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of the reagent</td>
			<td style="width:183px;">Company</td>
			<td style="width:123px;">Catalogue number</td>
			<td style="width:564px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

12.8K 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

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

マウス用ダーク/ライト遷移テスト

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

マイコプラズマの最適化されたPCRベースの検出

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

単離ミトコンドリアマウス心臓における透水性遷移孔の開口部のマルチパラメータ測定

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

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 ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

代替プロモーターの使用およびマウスBcrp1の転写調節を発見する方法

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM分析のための低細胞数とサンプル調製のための代替的アプローチ

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

成長因子 β シグナルを変換することによって誘導される血管内皮間葉移行の分子解析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...