Name: dna electroporation, isolation and imaging of myofibers
Uploaded: 2015-12-23T15:00:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about DNA Electroporation, Isolation and Imaging of Myofibers at JoVE.com

この作品は、国立衛生研究所NS047726、NS072027、およびAR052646を付与することによってサポートされていました。

この研究における方法は、医療施設内動物管理使用委員会（IACUC）のノースウェスタン大学フェインバーグ学校と倫理に従って行ったガイドラインを承認しました。すべての努力は苦痛を最小限にするために行われました。 マウスにおける屈筋Digitorumブレビスの in vivoエレクトロポレーション（FDB）筋肉バンドル1.実験手順<ol><li>哺乳動物細胞で発現することが知られているプロモーターを用いたin vivo発現のためにデザインプラスミド（例えば、サイトメガロウイルス、CMV）または筋肉内（例えば、筋肉クレアチンキナーゼ、MCK）6,7。大きなプラスミドは、低いレベルで発現することができます。 CMVプロモーターは6,7広く使用されてきました。 </li><li> 大腸菌からのプラスミドを精製大腸菌は、製造者の指示に従ってエンドトキシンを含まない条件を使用して。 5μgのプラスミド/μL - 2で滅菌、エンドトキシンを含まないTE緩衝液中のプラスミドを溶かします。 </li><li> concentratioにプラスミドを希釈し、分取滅菌エンドトキシンフリーのTE（2μgの/μl）を10μlの中のDNAの20μgのn個の滅菌マイクロチューブに注入ごとにバッファリング。注射当たり1つのアリコートを準備します。 </li><li> 8単位の最終濃度を与えるカルシウムとマグネシウムを含まない滅菌リン酸緩衝化生理食塩水で1Xにヒアルロニダーゼの株式（ - / - PBS）を希釈します。滅菌マイクロチューブに小分け注射当たり希釈ヒアルロニダーゼを10μl。 </li><li>鎮静さとテールやつま先のピンチに反応しないまで、2.5％イソフルランを用いてマウスを麻酔。正常な体温を維持するために加熱パッドや加熱テーブルの上に仰臥位で動物を置きます。 </li><li>予備校技術針サイトから発信し、外側に放射を使用して、ポビドンヨードスクラブとアルコールの交互のアプリケーションで三回マウスのフットパッドを清掃してください。この手法は、病原体の導入を最小限に抑え、無菌状態で針サイトを維持しています。 </li><li>目のフットパッドを注入皮膚や滅菌1mlシリンジを使用して筋肉の間に1倍のヒアルロニダーゼ溶液10μl（2 mg / mlの= 8台）付きの電子マウス。ヒアルロニダーゼは、筋線維へのプラスミドのより効率的な入力を可能にするために、細胞外マトリックスの成分を消化。ヒールの底に入力し、つま先に向かって針を進めます。針が後退するようにゆっくり液体をリリース。皮膚の下に注射部位を拡張し、ピンクをオンに開始します。 </li><li>繰り返しは反対の足で1.6と1.7を繰り返します。 </li><li>麻酔を外し、戻ってケージにマウスを置きます。マウスが完全に麻酔から回復できるようにします。 </li><li> 2時間待ちます。 </li><li>麻酔と足​​滅菌手順を繰り返して、1.5と1.6を繰り返します。 </li><li>ヒアルロニダーゼと同じやり方でフットパッドに希釈されたプラスミドDNAを注入します。最大容量は20μL以下であることを確認してください。デュアルプラスミド発現のために20＆の最大容量と各プラスミド20μgのを注入＃181; l以下。 </li><li>反対側の足蹠にプラスミド注射を繰り返すか、コントロール注射のための反対側の足を使用しています。 </li><li>最初に注射した足で、細かいピンセットで離れ下線筋肉から皮膚を持ち上げ、足の癒しつま先のラインに垂直位置につま先近くの皮膚のボールを介して1 27 G針を配置します。第二滅菌27 G針を取り、かかとの皮膚を水平に配置します。置き針は互いに離れ約1 cmの平行。 </li><li>ワニクランプを使用して、針が互いに接触していないことを確認することパラレル針に電極を固定します。粘土の使用は、所定の位置にクランプを固定することができます。 </li><li>電気刺激に電極を接続します。 </li><li> 100 V / cmので、1 Hzで、20パルス、持続時間が20ミリ秒/各を適用することにより、筋肉をエレクトロポレーション。つま先が刺激とけいれんがあります。 </li><li>必要に応じて反対側の足に刺激手順を繰り返します。 </li><li>針を外します。ポビドンヨードスクラブとフットパッドを拭きます。 </li><li>麻酔をオフにします。回復ケージにマウスを返すとアクティビティを監視します。予想通りの手順を実施した場合、動物は、30分以内に正常な歩行を取り戻し、プレ噴射の動作から歩行や活動レベルに差を示さないはずです。したがって、後手順の鎮痛剤は必要ありません。回復時には、動物施設に動物を返します。 注：タンパク質の発現は、エレクトロポレーション後早くも48時間のようにアッセイすることができます。 14日エレクトロポレーション10後-しかし、エレクトロポレーション後のより完全な回復を可能にするために、我々は7筋線維の単離および研究を好みます。発現は、4週間後にエレクトロポレーション限りアッセイすることができます。 </li></ol>屈筋Digitorumブレビス（FDB）繊維の単離のための2.実験方法<ol><li>コラゲナーゼを解凍します。各FDBバンドルの12ウェル組織培養プレート中に2個のウェルを準備します。 1でよく1メートルを追加予熱したダルベッコ改変イーグル培地+ウシ血清アルブミン（DMEM + BSA）のLおよび他にも予め温めておいたリンガー溶液1mlを加えます。また、料理をシルガード35ミリメートルに1×リンゲル液の新鮮な10ミリリットルを追加します。 </li><li>死を確実にするために、頸椎脱臼または他の方法に続いて、CO 2や麻酔ガスの吸入を介して動物を生け贄に捧げます。 </li><li>きれいなカミソリの刃を使用して足関節以上の後足を外し、35ミリメートルシルガード皿に1×リンゲル液に浸します。 </li><li>片足タイトピンは、5つま先の先端の各々を通ると足首で30 G針でシルガード皿に、唯一の上を向いて。 </li><li>足首から開始して、注意しないニックに、まっすぐつま先に向かって生え際に沿って足の側まで皮膚の下の筋肉を皮膚を切り取ります。足の反対側に繰り返します。 </li><li>細かいピンセットで足首の基部に皮を持ち、かかと全体の皮膚を切りました。 </li><li>肌をリフティング皮膚にないようニック筋肉に向かってあなたのはさみを指して、鉗子でフラップ。効果的に皮膚を削除し、下にある結合組織をスニップ。 </li><li> FDB筋バンドルを削除するには、骨から腱を解放するためにかかとで大きな腱を切りました。鉗子で腱を持ち、慎重にはさみでFDBに接続されているすべての結合組織を取り除く大きな白い腱からFDBバンドルを分析。適切に行われている場合、バンドルは、簡単に個々の数字につながる明るい白の腱を明らかに根本的な腱のリフトオフます。足からバンドルを解放つま先に近い腱でFDBをカットします。 </li><li>よく含有するDMEM + BSA中で全体の筋束を配置します。 </li><li>反対側の足の上に繰り返します。 </li><li>一旦全てのFDB筋を含む各ウェルのDMEM + BSAおよび筋肉にコラゲナーゼの100μlを添加、分離されています。 </li><li>消化前に倒立蛍光顕微鏡上のエレクトロポレーションの成功を確認してください。 properl行われた場合Y、90％のトランスフェクション効率の上方を達成することができます。 </li><li>約1時間、10％CO 2の加湿、37℃のインキュベーター内でプレートをインキュベートします。インキュベーション時間は、疾患モデルと背景とコラゲナーゼのロットに依存して変化し得ます。 </li><li> 1時間後、慎重に含むウェルのみリンゲル液にFDBバンドルを転送します。 </li><li> 1ミリリットルピペットを用いてリンゲル液で十分にFDBバンドルを粉砕します。バンドルを簡単にピペットの先端を通過することができるようにきれいなかみそりの刃でピペットチップの先端をカットします。静かにバンドルが渡すと、先端に平行ダウンすることができ、筋肉（〜15倍）を粉砕​​します。無傷の繊維は、リンゲル液中に落ちます。繊維が簡単にバンドルをオフに該当しない場合には、コラゲナーゼ溶液に背筋を配置します。 </li><li>プレート当たり〜500μlの皿の上の孤立した繊維をプレート。 </li><li>筋肉を15分間ウェル中で消化できるようにします。 </li><li>繰り返し2.15ステップ2.17にバンドルのサイズが小さくなるまで、繊維の大部分は、バンドル（2〜3回の通常合計）から解離されます。一度筋肉は簡単に1ミリリットルピペットチップを通過し解離しました。 </li><li>繊維が15分の最低皿に添付してみましょう。新鮮なリンゲル液は、残留コラゲナーゼを希釈し、プレートに添加し、または時間的制約に応じて変更することができます。 注：繊維は今イメージングのための準備ができています。 </li></ol>共焦点顕微鏡を用いて、レーザ誘起膜損傷の可視化のための3実験手順<ol><li>リンゲル液にFM 4-64染料2.5μMのFM 1-43の300μlの画像化の直前にロード繊維。一般的に、分離後の画像繊維15分に2時間。しかし、繊維は、24時間後の単離まで撮像することができます。 </li><li> UVレーザおよび60X / 63Xの対物レンズを搭載した共焦点顕微鏡のガラス底皿を置きます。 </li><li>繊維の位置を確認します。繊維がしっかりと接続されていることを確認顕微鏡ベースをタップして皿に。解離過程で破損された繊維を除外します。損傷を受けた繊維が強くカールまたは屈曲膜ブレブ、FM色素の取り込みを高レベルで含有してもよいです。 </li><li>繊維上の膜損傷を誘導するために、核の明確な視野を持つ撮像窓の中央に筋鞘を置きます。筋細胞膜が非常にシャープであるように、FM色素チャンネルを使用して、筋細胞膜に焦点を当てています。 </li><li> FM 4-64チャネルを利用し、筋細胞膜上のROIクロス髪を置きます。 </li><li> 3秒（X〜350nmの領域350 nm）の80％の電力で1画素点を照射します。 FM色素は、損傷部位に細胞に入ります。電力及び時間は傷害の面積を増加または減少させるように調整することができます。 </li><li>最大5分ごとに10秒間、その後、損傷時には、損傷前に2秒ごとポスト損傷を繊維の画像をキャプチャします。タイミングは、必要に応じて調整することができます。個々の画像はイメージJ.にタイムラプスムービーに変換することができます</li><li>手順を繰り返し皿ごとに最大3繊維上の3.7を介して3.3。 </li></ol>

<ol>
	<li>Blau, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+differentiated+state.">Plasticity of the differentiated state.</a> Science. 230, 758-766 (1985).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270, 725-727 (1977).</li><li>Richler, C., Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+cultivation+and+differentiation+capacities+of+myogenic+cell+lines.">The in vitro cultivation and differentiation capacities of myogenic cell lines.</a> Dev Biol. 23, 1-22 (1970).</li><li>Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+differentiation+potentialities+during+prolonged+cultivation+of+myogenic+cells.">Retention of differentiation potentialities during prolonged cultivation of myogenic cells.</a> Proc Natl Acad Sci USA. 61, 477-483 (1968).</li><li>Mamchaoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+pathological+human+myoblasts:+towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.</a> Skelet muscle. 1, 34 (2011).</li><li>DiFranco, M., Quinonez, M., Capote, J., Vergara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transfection+of+mammalian+skeletal+muscles+using+in+vivo+electroporation.">DNA transfection of mammalian skeletal muscles using in vivo electroporation.</a> J Vis Exp: JoVE. , (2009).</li><li>Swaggart, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+A6+modifies+muscular+dystrophy+by+mediating+sarcolemmal+repair.">Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.</a> Proc Natl Acad Sci USA. 111, 6004-6009 (2014).</li><li>Kerr, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlin+stabilizes+stress-induced+Ca2++signaling+in+the+transverse+tubule+membrane.">Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.</a> Proc Natl Acad Sci USA. 110, 20831-20836 (2013).</li><li>Anderson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micropeptide+encoded+by+a+putative+long+noncoding+RNA+regulates+muscle+performance.">A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.</a> Cell. 160, 595-606 (2015).</li><li>Roche, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+histological+changes+in+skeletal+muscle+following+in+vivo+gene+transfer+by+electroporation.">Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation.</a> Am J Physiol Cell Physiol. 301, C1239-1250 (2011).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca(2+)-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes.</a> Cell. 106, 157-169 (2001).</li><li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+blebbing+as+an+assessment+of+functional+rescue+of+dysferlin-deficient+human+myotubes+via+nonsense+suppression.">Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression.</a> J Appl physiol. 109, 901-905 (2010).</li><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MG53+nucleates+assembly+of+cell+membrane+repair+machinery.">MG53 nucleates assembly of cell membrane repair machinery.</a> Nat Cell Biol. 11, 56-64 (2009).</li><li>Marg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+repair+is+a+slow+process+and+includes+EHD2.">Sarcolemmal repair is a slow process and includes EHD2.</a> Traffic. 13, 1286-1294 (2012).</li><li>Bansal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li><li>Defour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlin+regulates+cell+membrane+repair+by+facilitating+injury-triggered+acid+sphingomyelinase+secretion.">Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion.</a> Cell Death Dis. 5, e1306 (2014).</li></ol>

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生体内での蛍光タグ付きタンパク質を研究するためのエレクトロポレーションの使用は忠実に筋肉の全体のセル構造を複製し、適切な細胞株モデルの欠如与えられた筋肉の研究のために理想的に適しています。このプロトコルは、エレクトロポレーションおよびレーザー創傷後のタンパク質の局在化及び転座の詳細な画像を提供するために、高解像度の共焦点顕微鏡の利用を説明して...

個々の筋線維は、大規模な、高度に組織化された合胞体細胞です。筋肉のためのいくつかの細胞培養モデルがあります。しかし、これらのモデルは、彼らが完全に成熟した筋線維に分化していない彼らの主要な制限として持ちます。例えば、C2C12およびL6細胞株は、マウス、ラット、それぞれ1-4から誘導されます。血清飢餓の条件下で、単核「筋芽細胞様」細胞は、増殖を止める細胞周期の撤退を受けて、サルコメアと多核細胞を形成する筋形成プログラムに入力して、筋管と呼ばれます。長期培養条件では、筋管が収縮特性を示すことができ、培養中の「けいれん」。ヒト細胞株は、今も5確立されています。これらの不死化細胞株に加えて、単核筋芽細胞が筋から単離することができ、血清飢餓の同様の条件下での筋管を形成します。これらの細胞株および初代筋芽細胞培養物は、高度に使用されていますそれらはプラスミドでトランスフェクトまたはウイルスで形質導入し、基本的な細胞生物学的プロセスを研究するために使用することができるので、FUL。筋管を形成するように誘導された場合しかし、これらの細胞は、さらに、成熟した筋組織の顕著な特徴の多くを欠いています。具体的には、筋管は、個々の成熟した筋線維よりもはるかに小さく、筋線維の正常な形状を欠いています。批判的に、筋管は、筋形質全体で効率的なのCa 2+放出のために必要な膜状のネットワークを横断（T-）細管を欠いています。 一次筋芽細胞または筋原細胞株の代替方法は、成熟した筋線維を使用することを伴います。形質転換は、標識タンパク質の発現を確立するために使用することができるが、この方法は、コストと時間がかかる。マウス筋肉のインビボ電気穿孔は、速度と信頼性6-10ための好ましい方法として浮上しています。 in vivoでのエレクトロポレーションおよび筋線維ヘクタールを効率的に分離するための方法マウス短趾屈筋（FDB）の筋肉6のために最適化されまし。方法は簡単に完了し、プラスミドからin vivo発現誘導するために最小限の侵襲性ですることができます。このアプローチは現在、筋細胞膜6,7のレーザ破壊後のイメージングを含む高解像度イメージング法と組み合わされます。蛍光色素および蛍光標識されたタンパク質の発現の組み合わせは、成熟筋線維中の細胞の生物学的プロセスをモニターするために使用することができます。

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>11995-073</td><td>Dissection Media&lt;br/&gt; Dilution: 50 ml +100 g BSA</td></tr><tr><td>Collagenase, Type II</td><td>Life Technologies</td><td>17101-015</td><td>Fiber Digestion&lt;br/&gt; Dilution: 160 mg / 4 ml DMEM (stock 40 mg/ml aliquot)</td></tr><tr><td>Falcon 12-well dishes</td><td>Fisher Scientific</td><td>877229</td><td>Fiber Digestion</td></tr><tr><td>Ringers Solution</td><td></td><td></td><td>Fiber Digestion and Imaging&lt;br/&gt; Dilution: 146 mM NaCl&lt;br/&gt; 5 mM&amp;nbsp;&amp;nbsp; KCl&lt;br/&gt; 2 mM&amp;nbsp;&amp;nbsp; CaCl2&lt;br/&gt; 1 mM&amp;nbsp;&amp;nbsp; MCl2&lt;br/&gt; 10 mM HEPES&lt;br/&gt; pH7.4 in H2O</td></tr><tr><td>1 ml Pipettes</td><td>Axygen</td><td>T-1000-C-R</td><td>Digestion</td></tr><tr><td>Razor Blades</td><td>Personna</td><td>94-120-71</td><td>Digestion</td></tr><tr><td>Precision Glide 30 G needle</td><td>BD Biosciences</td><td>305128</td><td>Dissection</td></tr><tr><td>1x PBS (-/-)</td><td>Life Technologies</td><td>14190-250</td><td>Dissection</td></tr><tr><td>Sylgard 184</td><td>Fisher Scientific</td><td>50-366-794</td><td>Dissection</td></tr><tr><td>Forceps</td><td>FST by Dumont</td><td>#5/45</td><td>Dissection</td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-4913</td><td>Dissection</td></tr><tr><td>Scissors</td><td>World Precision Instruments</td><td>500260</td><td>Dissection</td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td>Dissection media&lt;br/&gt; Dilution: 100 mg/50 ml DMEM</td></tr><tr><td>Falcon 60 mm dishes</td><td>Fisher Scientific</td><td>08772B</td><td>Dissection- used to hold sylgard</td></tr><tr><td>Clay</td><td></td><td></td><td>Electroporation</td></tr><tr><td>Stimulator&amp;nbsp;</td><td>Grass</td><td>s88x</td><td>Electroporation</td></tr><tr><td>Clamps</td><td>Radio Shack</td><td>270-356</td><td>Electroporation</td></tr><tr><td>Triadine</td><td>Aplicare</td><td>82-220</td><td>Electroporation</td></tr><tr><td>Precision Glide 27 G needle</td><td>BD Biosciences</td><td>305136</td><td>Electroporation</td></tr><tr><td>Simulator</td><td>Grass</td><td>SIU-V</td><td>Electroporation</td></tr><tr><td>Gauze</td><td>Covidien</td><td>2146</td><td>Electroporation</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H4272</td><td>Injection&lt;br/&gt; Dilution: Add 160 ul PBS (1x) to 40 &amp;micro;l 5x stock</td></tr><tr><td>1 cc U-100 Insulin Syringe (28G1/2)&amp;nbsp;</td><td>BD Biosciences</td><td>329420</td><td>Injection</td></tr><tr><td>Eppendorf Tubes</td><td>Fisher Scientific</td><td>02-682-550</td><td>Injection</td></tr><tr><td>35 mm glass bottom Microwell dish</td><td>MaTek</td><td>P35G-1.5-14-C</td><td>Microscopy</td></tr><tr><td>FM 4-64</td><td>Life Technologies</td><td>T-13320</td><td>Microscopy&lt;br/&gt; Dilution: Final 2.5 ug/ml</td></tr><tr><td>FM 1-43</td><td>Life Technologies</td><td>T-35356</td><td>Microscopy&lt;br/&gt; Dilution: Final 2.5 ug/ml</td></tr><tr><td>Endotoxinfree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Plasmid Purification</td></tr></tbody></table>

dna electroporation, isolation and imaging of myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

エレクトロポレーションは、効率的に短趾屈筋（FDB）の筋肉の束（ 図1A）に精製したプラスミドDNAを導入して低侵襲的手法です。七日には、蛍光標識タンパク質が分離された筋線維（ 図1B）で可視化され、トランスフェクション後。単離された繊維は、次に、播種し、共焦点顕微鏡で画像化のために調製されます。繊維は、レーザ誘起損傷を...

Watch this Scientific Journal Video about DNA Electroporation, Isolation and Imaging of Myofibers at JoVE.com

DNA Electroporation, Isolation and Imaging of Myofibers

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNAエレクトロポレーション、筋線維の単離およびイメージング

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

dna-electroporation-isolation-and-imaging-of-myofibers

Research

JoVE Journal

Biology

12.2K ビュー.  Northwestern University. This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure. 

ビデオ: DNA Electroporation, Isolation and Imaging of Myofibers

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

神経筋障害の治療法を調査するヒト線維芽細胞の筋芽細胞への直接リプログラミング

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

遺伝学

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

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

低侵襲の筋肉 (MIME) の埋め込み - ドナー細胞の筋を容易にする新しい実験手法

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

医学

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

解離調製物から胚および幼虫のゼブラフィッシュ骨格筋線維の分析

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

出生後初期の発達におけるエメリー・ドレイフス型筋ジストロフィーのマウスモデルからの単一の筋繊維の分離と培養

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

筋芽細胞の分化と融合中にトラック核へライブ イメージングを利用しました。

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

骨格筋生検からミオフィブリルを分離し、ナノニュートン分解力トランスデューサーによる収縮機能を決定する

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

成人筋幹細胞機能評価のための単一筋繊維培養アッセイ Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

可変長マウス筋線維の酵素的単離と特性評価

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

著者スポットライト:線維タイプ間の興奮収縮結合を研究するためのマウス後肢筋からの長筋線維の分離

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

マウスの6つの後肢筋の酵素的解離によって得られた無傷の短、中、および長の骨格筋線維:指屈筋を超えて

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

DNAエレクトロポレーション、筋線維の単離およびイメージング

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神経筋障害の治療法を調査するヒト線維芽細胞の筋芽細胞への直接リプログラミング

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成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

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出生後初期の発達におけるエメリー・ドレイフス型筋ジストロフィーのマウスモデルからの単一の筋繊維の分離と培養

筋芽細胞の分化と融合中にトラック核へライブイメージングを利用しました。

骨格筋生検からミオフィブリルを分離し、ナノニュートン分解力トランスデューサーによる収縮機能を決定する

成人筋幹細胞機能評価のための単一筋繊維培養アッセイ Ex Vivo

マウスの6つの後肢筋の酵素的解離によって得られた無傷の短、中、および長の骨格筋線維:指屈筋を超えて

DNAエレクトロポレーション、筋線維の単離およびイメージング

要約

さらに動画を探す

この動画の章

神経筋障害の治療法を調査するヒト線維芽細胞の筋芽細胞への直接リプログラミング

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

低侵襲の筋肉 (MIME) の埋め込み - ドナー細胞の筋を容易にする新しい実験手法

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

解離調製物から胚および幼虫のゼブラフィッシュ骨格筋線維の分析

出生後初期の発達におけるエメリー・ドレイフス型筋ジストロフィーのマウスモデルからの単一の筋繊維の分離と培養

筋芽細胞の分化と融合中にトラック核へライブ イメージングを利用しました。

骨格筋生検からミオフィブリルを分離し、ナノニュートン分解力トランスデューサーによる収縮機能を決定する

成人筋幹細胞機能評価のための単一筋繊維培養アッセイ Ex Vivo

マウスの6つの後肢筋の酵素的解離によって得られた無傷の短、中、および長の骨格筋線維:指屈筋を超えて

筋芽細胞の分化と融合中にトラック核へライブイメージングを利用しました。