オリジナルのGFP - PEM1は博士レイリーから寄贈されたもの。この作品は、NIHとエリソン医学財団からVGへの補助金によってと同様にサポートされて

このプロトコルでは、DSBのが一過性発現珍しい切断エンドヌクレアーゼI - SCEI 3によって生体内で誘導される1,2を構築する染色体に統合されたレポーターにDNA DSB修復の分析のための方法を説明します。統合された分析は、染色体のコンテキスト内でDSBの修復を分析することの利点を提供します。しかし、このプロトコルは、初代培養細胞を扱うとき問題が発生することが継長時間細胞を、必要になります。 別のアプローチは、レポーター構築物はI - SCEIまたはをHindIIIエンドヌクレアーゼでin vitroで消化し ​​、直鎖DNAとして細胞にトランスフェクトされている体外アッセイである。 exctrachromosomalアッセイプロトコール4次の修正で以下に説明する統合されたアッセイに似ています。体外アッセイ用レポーターコンストラクトの統合を省略し、代わりにI - SCEIまたはHindIIIとトランスフェクションのコントロールとしてpDsRed2 - N10.1μgのin vitro で直線化プラスミドNHEJのレポーター0.5μgの細胞を同時トランスフェクション。 HRアッセイ用プラスミドの共トランスフェ2μgのHRの記者は、I - SCEIまたはHindIIIと0.1μgのpDsRed2 - N1で直鎖状。三日間トランスフェクション後にFACSで細胞を分析する。体外アッセイは、継代し、複数の細胞株の解析を容易にする豊富なセルを回避し、一次分離株におけるDSB修復の解析が可能になります。 1。 NHEJとHRのレポーターコンストラクト NHEJのレポーターカセット5 PEM1遺伝子（GFP - PEM1）から工学3キロバイトイントロンを持つGFPの遺伝子が含まれています。 PEM1イントロンは、DSBが誘導のためのHindIII及びI - SCEIエンドヌクレアーゼ（図1a）のための認識配列に挟まれたアデノウイルスのエクソンが含まれています。 I - SCEIのサイトでは、逆方向になります。 I - SCEIはnonpalindromic認識配列を有し、したがって2つの逆方向のサイトには互換性のDNA末端（図1c）を生成します。互換性のDNAは、最高の自然に発生するDSBのを模倣終了します。無傷NHEJカセットはGFPアデノウイルスエクソンGFP ORFを破壊するように負である。 I - SCEIによるDSBが誘導されると、アデノウイルスエクソンは削除され、NHEJは、GFPの遺伝子（図2）の機能が復元されている。このNHEJレポーターのユニークな特徴は、イントロンが欠失および挿入を許容することができるので、それはNHEJイベントの広いスペクトルを検出できることです。 HRレポーターカセット1（図1b）もGFP - PEM1に基づいています。 HRのカセットでは、GFP - PEM1の第一エクソンは、3つの制限部位、I-SceI/HindIII/I-SceIの挿入と組み合わせて22 bpの欠失を含んでいます。欠失は、GFPがNHEJのイベントで再構成することが不可能になります。つのI - SCEIのサイトでは、I - SCEIの消化は、互換性のない両端（図1c）を残すように、逆方向になります。 GFP - PEM1の最初のコピーは、GFP - PEM1のpromoter-less/ATG-less第一エクソンとイントロンが続いている。無傷の構造はGFP陰性である。 I - SCEIの消化によるDSBの誘導時に機能的なGFPの遺伝子は、最初にGFP - PEM1エクソンの二つ変異のコピーの間に分子内や分子間遺伝子変換によって再構成される。 GFP遺伝子の番目のコピーは、最初のATGコドンと第2エキソンを欠くクロスオーバーまたは一本鎖アニーリングされているためとGFPの活動を復元しません。この設計は、哺乳動物細胞で優勢なHRの経路（図3）である遺伝子変換の排他的な検出、することができます。 2。レポーターの統合は、ゲノムに構築<ol><li> NHEJまたはHRのレポータープラスミドの高品質な準備を行う。最良の結果を得るにはキアゲン遠藤フリーキットを使用してください。 280分の260 nmでの吸光度がプラスミドの濃度と純度を測定します。 </li><li> 50μlのNheIを50 U（反応の合計量）37で6時間で消化することにより、レポータープラスミド10μgを線形℃のインキュベーター（蒸発を防ぐために、乾燥したブロックを使用しないでください）​​。キアゲンQiaexIIキット、10mMトリス- HCl pH8.0を20μlのに溶出したDNAを用いた消化液からDNAを精製する。 280分の260 nmの吸光度でDNA濃度と純度を測定します。通常は、精製の間にDNAの20〜30％を失うことになる。未消化のレポーターコンストラクトと一緒に、ゲルに小さな分量（2μl）を実行して、収量と消化を確認してください。精製された線形化レポーター構築物を20℃で保存することができます</li><li>トランスフェクションのための準備で、最高の生育状態に細胞​​をもたらす。凍結バイアルから開始する場合、トランスフェクションの前に細胞を2回に分割。コンフルエント細胞のプレートから始まる場合、それらを一度に分割。 </li><li> 70〜80％コンフルエント（通常正常ヒト線維芽細胞のための二日間、メッキ5 × 10 5 cells/100 mmのプレートなら、）に細胞を成長させる。それは、対数増殖に細胞を持って最高のトランスフェクション効率のための非常に重要です。活発に成長している文化は、Mステージの細胞を（表面に付着した丸みを帯びた細胞として見られる）が含まれています。 </li><li> TRANSFAmaxa Nucleofector以下のメーカーの推奨を用いて細胞をECT。線維芽細胞は、NHDFのソリューションとプログラムのU20を使用してください。トランスフェクションのために、2つの100 mmプレート（〜2 × 10 6個の細胞）から細胞を採取する。それは上の細胞をトリプシン処理することではないが重要です。とすぐにプレートから切り離された細胞の80％として、トリプシン処理を停止します。 NHDF溶液中で細胞を再懸濁します。細胞がNHDFのソリューションに敏感である、従って、インキュベーション時間を最小限に抑え、穏やかに細胞を混在させること。線形化されたレポーター構築0.5μgので細胞をトランスフェクション。レポーターの単一コピーの統合における正常ヒト線維芽細胞の結果で使用されるこれらのトランスフェクション条件は、integrantsの大半を構築する。 </li><li>トランスフェクション後に1 mg / mLのジェネティシン（G418）24を追加することによって、染色体に統合されたレポーターコンストラクトを持つセルを選択します。 7〜10日の選択を続行します。 </li><li> G418耐性コロニーを（あなたが個々のコロニーやプール耐性クローンを選択できます）選択。約5 100 mmのプレートに文化を展開します。将来の使用のための3つのプレートを凍結し、所望の細胞数（通常10百ミリメートルプレートに5つのトランスフェクションのために十分である）に、残りの二つのプレートを展開します。 </li><li>細胞は70〜80％コンフルエント（2日間線維芽細胞を100 mmプレート当り5 × 10 5細胞を播種した後に）到達すると、細胞はI - SCEIが発現するプラスミドでトランスフェクトする準備が整いました。 </li></ol> 3。 I - SCEIエンドヌクレアーゼの一過性発現によるDSBの誘導<ol><li>トランスフェクション〜5μgのI - SCEI発現プラスミドおよび0.1μgのAmaxa Nucleofectorを（NHDFのソリューションおよび線維芽細胞のためのU20プログラムを使用して使用してトランスフェクション効率の制御などのpDsRed2 - N1（Clontech社製）の混合物と対数的に成長する文化の2 × 10 6個の細胞（二つのプレート） ）。 </li><li>同時に、（i）の5μgのプラスミドEGFP発現で〜2 × 10 6個の細胞をtrasfectingによってFACSための3つのキャリブレーションコントロールを用意すること、（ii）5μgのpDsRed2 - N1、表現していないコントロールプラスミドの（ⅲ）5μgを蛍光タンパク質（我々がコードするプラスミドHPRTミニ遺伝子を使用している）。 </li><li> GFPとDsRedタンパク質の発現に十分な時間を提供するために、四日間のために細胞を成長させる。 </li><li> GFPとDsRedの検出のためのフィルターと蛍光倒立顕微鏡でGFPとDsRedタンパク質のトランスフェクションと発現の効率をトランスフェクションを確認した後（省略）三日。 </li></ol> 4。フローサイトメトリーによるGFP +およびDsRed +細胞の解析<ol><li>トランスフェクションの収穫I - SCEIトランスフェクトした細胞とコントロール細胞の後の四日間。 PBS500μlに細胞を再懸濁し、FACSチューブに細胞を移す。この段階ではすべての細胞を回収し、丸い形の細胞を持つことが重要です。これを達成するために、それは長いトリプシン処理の時間またはより強力なトリプシンを使用することをお勧めします。細胞の99％がプレートから切り離されていることを確認し、顕微鏡下で細胞を観察することによって丸い形を持っている。 （箔と氷のバケツをカバーする）、光から保護氷上で細胞を維持する。数時間のために氷上で細胞を保存することが可能です。のために最良の結果は、収穫直後の細胞を分析する。 </li><li> GFP、DsRedを、およびネガティブコントロールとFACSをキャリブレーション。分析のすべての蛍光細胞（図4）を含めるために、電圧と色補正を調整します。実験試料の蛍光強度は、対照のそれよりも低くなることに留意してください。 </li><li>分析I - SCEI FACSにより細胞をトランスフェクション。私たちは、それぞれの治療のために少なくとも20,000のセルをカウント。 GFPとDsRedの蛍光​​との間の潜在的な干渉を防止するためには、GFPのパーセント+またはDsRed + 25％以下の細胞を維持することが推奨されます。 DsRedを+またはGFP +細胞の割合が高い場合pDsRed2 - N1またはI - SCEIプラスミドの量を減らす。我々の実験では、唯一のpDsRed2 - N1プラスミドのではなく、プラスミドI - SCEIの量を調整する必要がありました。 </li><li> GFP +細胞のパーセントは、DNA DSB修復の効率に相当するとDsRed +細胞のパーセントは、トランスフェクションの効率を示します。 DsRedタンパク質+細胞へのGFP +細胞の比率としてDNA DSB修復の相対的な効率を算出する。 </li><li>修理終了の接合は、修復の精度をテストするために配列決定することができる。レポーターコンストラクトは、複製の大腸菌の起源とカナマイシン耐性遺伝子（図1）を含んでいます。これは、EcoRI酵素、再環状化、および有能な大腸菌細胞への転換でゲノムDNAを消化することによって構造を救出することができます。救出プラスミドを制限酵素消化および配列決定により分析されています。 </li></ol> 5。代表的な結果コントロールのトランスフェクションのとNHEJとHRの実験の代表的なFACSの結果を図4と図5に示す。蛍光強度と蛍光細胞の数は、を積分を含むI - SCEIトランスフェクション細胞で低くなります。ED NHEJとHRの構造（図5）GFPとこれらの細胞にDsRedの構造のコピー数の違いによるコントロール（図4）と比較。実験の各セルは、統合されたレポーター構築のコピーを1つだけ含まれている、したがって、修復が成功のイベントには、細胞の割合で再構築GFP遺伝子を務める。ヒト線維芽細胞では0.1μgpDsRed2 - N1のトランスフェクションは細胞あたり約1プラスミドのコピーを提供します。 NHEJとHRの効率は、GFP + / DsRedタンパク質+細胞の比率として計算されます。 NHEJは、2つのヒト細胞におけるHRよりも効率的なプロセスです。ヒト線維芽細胞ではNHEJの効率は一般的に0.6から1.3であり、そしてHRの効率は0.05から0.3です。 DSB効率は比のGFP + / DsRedを+ I - SCEIとDsRed2の- N1の混合物中の細胞の変化として測定されるためのプラスミドは、結果に影響を与える可能性があります。プラスミドの品質はまた、トランスフェクション効率を変更することにより、結果に影響を与える可能性があります。したがって、一貫性のある測定値を得るために、それは一つの実験内の同じプラスミドミックスを使用することが重要です。また、DSBの修復効率は、細胞型、細胞周期の段階、および統合された構造の染色体位置の影響を受けます。 <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="図1" /> 図1。レポーターはNHEJ（）とHR（B）によるDNA DSB修復の分析のために構築する。 （C）互換性のないDNAは、2つの逆方向のI - SCEIのサイトでの消化によって生成された終了 SD、スプライスドナー、SA、アクセプタースプライス、日陰の正方形、ポリアデニル化部位、ネオ/仮名、単一のORF 2つのプロモーターによって制御される：。哺乳類におけるSV40付与するネオマイシン耐性細胞、及び大腸菌のβ-ラクタマーゼ付与kanamicyn抵抗、オリ、E.大腸菌のpUC複製起点。 <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="図2" /> 図2。 。レポーターNHEJの分析のために構築しこの中でGFP遺伝子を構築は非アクティブですがDSBと成功NHEJの誘導時に構造物はGFPの+になります。次に示すのは、NHEJによってこのレポーターの修理の製品です。 <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="図3" /> 図3。レポーターは、遺伝子変換によるHRの分析のために構築する。遺伝子変換（GC）によってI - SCEI、HRによってDSBが誘導されると、アクティブなGFPの遺伝子を再構成。 GC、交差（クロスオーバー）、一本鎖（SSA）アニーリング、およびNHEJ：以下に示す主要なDNA修復経路により、このレポーターの修復の製品です。 X -オーバー修理製品は分子内組換えのために示されている、exramolecular組換えは、同じ製品を与えるが、1つの染色体上に位置される。アクティブなGFPタンパク質を発現する遺伝子（GFP +）緑の色で示されている。 <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="図4" /> 図4。 FACS解析のためのパラメータのキャリブレーション。自動蛍光細胞を除くためのネガティブコントロールとして使用されるプラスミドpHPRT、でトランスフェクション（）線維芽細胞。 （B）繊維芽細胞は、GFP +細胞（緑色の領域）のためのゲートを設定するために使用される、プラスミドpEGFPをトランスフェクト。 （C）線維芽細胞はDsRedタンパク質+細胞（赤色の領域）のためのゲートを設定するために使用されるプラスミドpDsRed2 - N1でトランスフェクト。 <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="図5" /> 図5。染色体に統合されたレポーターコンストラクトと正常ヒト線維芽細胞におけるNHEJ（）とHR（B）の分析の典型的な結果。

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

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蛍光NHEJとHRのレポーターアッセイでは、別々にin vivoで各DSBの修復経路を測定するための定量的な方法を提供します。 FACSは、確実に20,000細胞で10 GFP +細胞を検出することができるようなアッセイは、非常に敏感です。アッセイは、DSBの2の誘導後数分または数時間以内GFP +細胞の出現を検出することにより、&quot;リ ​​アルタイム&quot;で修復イベントを測定するために適合させ?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
</tr>		</tbody>
		</table>
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analysis of dna double-strand break (dsb) repair in mammalian cells

DNA二本鎖切断は、遺伝情報と細胞死の巨大な損失につながる可能性が最も危険なDNAの損傷です。非相同端接合（NHEJ）と相同組換え（HR）：2つの主要な経路を用いて細胞の修復DSBが。 NHEJとHRの摂動は、しばしば早期老化と腫瘍形成に関連付けられている、それゆえに各DSBの修復経路を測定する定量的な方法を持つことが重要です。当研究室では、NHEJとHRの高感度かつ定量的な測定を可能にする蛍光レポーターコンストラクトを開発しました。構文は、DSBが誘導のための珍しいカットI - SCEIのエンドヌクレアーゼの認識部位を含む工学GFPの遺伝子に基づいています。 GFP遺伝子は、追加のエクソンによって、または変異により不活性化されるように始まる構造はGFP陰性である。 NHEJまたはHRによるI - SCEI誘起ブレイクの成功修復は、機能的なGFPの遺伝子を復元します。フローサイトメトリーによってカウントGFP陽性細胞の数は、NHEJまたはHR効率の定量的な尺度を提供する。

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

この記事では、GFPベースの蛍光を示して<em&gt; in vivoで</em&gt;別々に相同組換えを定量化し、非相同哺乳類細胞に結合する終了アッセイ。

哺乳動物細胞におけるDNA二重鎖切断（DSB）修復の解析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K ビュー.  University of Rochester. この記事では、GFPベースの蛍光を示して in vivoで別々に相同組換えを定量化し、非相同哺乳類細胞に結合する終了アッセイ。

ビデオ: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用して哺乳類の骨格筋のDNAトランスフェクションインビボでのエレクトロポレーション

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

生物学

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

解離成体哺乳類の神経細胞へのDNAの核内マイクロインジェクション

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

マイクロインジェクションによる哺乳動物細胞におけるmRNA核外輸送の速度論の解析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

哺乳動物細胞におけるヘム合成レベルの測定

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.