We thank Drs. Tom Cooper from the Baylor College of Medicine, Mani S. Mahadevan from the University of Wisconsin-Madison, and Didier Trono from the University of Geneva for reagents. This work is supported by a University of Houston startup fund (YL), American Heart Association grant (YL, 11SDG5260033), and the National Natural Science Foundation of China (XP, 81460047).

1. C2C12 Zellkultur <ol><li> Pflegen C2C12-Maus-Myoblasten in einer 100 mm-Platte in Wachstumsmedium (Dulbecco-modifiziertem Eagle-Medium (DMEM)), ergänzt mit 20% fötalem Rinderserum, 100 U / ml Penicillin, 100 ug / ml Streptomycin und 2 mM L-Glutamin. Lassen Sie C2C12 agierten Zellen etwa 50 werden - 60% konfluent. </li><li> Verwerfen das Wachstumsmedium und wasche C2C12 Zellen mit 3 ml Raumtemperatur phosphatgepufferter Kochsalzlösung (PBS). Entfernen Sie die PBS und fügen 500 ul 0,25% Trypsin-EDTA die Zellen zu lösen. Die Platte wird in einem 37 ° C, 5% CO 2 Inkubator 3 - 5 min. </li><li> Neutralisieren des Trypsins durch Zugabe von 3 ml Wachstumsmedium. Pipette 6 - 8 mal um die Zellen zu suspendieren. 1 ml suspendierter Zellen zu einer neuen 100 - mm - Platte und inkubiere bei 37 ° C, 5% CO 2. </li></ol> 2. C2C12 Zelltransfektion / Transduktion und Selektion <ol><li> C2C12 Plasmidtransfektion <ol><li> 24 Stunden vor der Transfektion, pspäten 7,0 x 10 5 C2C12 Zellen in eine Vertiefung einer Sechs-Well - Platte für jede Transfektion. Anmerkung: Dies führt zu einer 50 bis 60% Konfluenz durch den Zeitpunkt der Transfektion. </li><li> Lassen Sie das Transfektionsreagenz auf Raumtemperatur vor dem Gebrauch zu kommen. </li><li> In 2 ug Plasmid-DNA (GFP-CUG5 oder GFP-CUG200) in 200 &amp; mgr; l vorgewärmten serumfreien Medium und Wirbel kurz. In 6 ul Transfektionsreagenz auf das Medium und mischen sofort für 30 Sekunden einen Wirbel verwenden. Inkubieren der Mischung für 30 min bei Raumtemperatur. </li><li> Ändern Sie die Medien von C2C12-Zellen zu 2,5 ml Serum-freien Wachstumsmedium. Fügen Sie das Transfektionsgemisch zu den Zellen nach der Inkubation. Rück die Platten zu einer 5% CO 2, 37 ° C Inkubator. </li><li> Halten Sie die Zellen in Transfektionsgemischs / Serum-freien Wachstumsmedium für 4 Stunden oder bis über Nacht. Ändern Medien zurück zu den Wachstumsmedien am nächsten Tag oder wenn Zytotoxizität ist offensichtlich. Hinweis: Die Zytotoxizität wird beurteilt durch mikroskopische Untersuchung des cells. Beachten Sie die Veränderungen der Zellmorphologie und Zellablösung. Solange es keine offene Zytotoxizität ist, Inkubation über Nacht in serumfreiem Wachstumsmedium erhöht die Transfektionseffizienz. </li><li> Markieren Sie die Zellen 48 Stunden nach der Transfektion durch Zugabe von 1,2 bis 1,6 mg / ml G418 für ~ 10 Tage, bis die Kontrollkultur (ohne die Plasmide transfiziert) keine lebenden Zellen haben. Ändern Sie die Medien alle zwei Tage mit Wachstumsmedien mit G418 ergänzt. </li><li> Pflegen die G418 - resistenten Zellen in Wachstumsmedium und 5% CO 2 bei 37 ° C für nachfolgende Experimente. </li></ol></li><li> Retrovirus Vorbereitung und C2C12 retrovirale Transduktion <ol><li> Bereiten HEK 293 Kulturmedium durch DMEM mit hohem Glucose mit 10% fötalem Rinderserum ergänzt, 100 U / ml Penicillin, 100 ug / ml Streptomycin und 2 mM L-Glutamin. <ol><li> Etwa 24 Stunden nach der Transfektion vor, Platte 4,0 x 10 6 ekotropischen HEK 293-basierte Verpackungszellen in einer 100 - mm - Platte (80% Zusammenfluß bei Transfektion) containing Zellkulturmedium. </li></ol></li><li> Lassen Sie das Transfektionsreagenz auf Raumtemperatur vor dem Gebrauch zu kommen. </li><li> Mischen Sie 15 ug Plasmid-DNA (pMSCV-Puro oder pMSCV-Celf1Flag-Puro) in 1 ml Serum-freien Wachstumsmedium. Vortex kurz. In 30 ul Transfektionsreagenz aus Schritt 2.2.2 in die DNA / Serum-freien Wachstumsmedium Rohr und gut mischen Wirbel verwenden. Inkubieren der Mischung bei Raumtemperatur für 30 min. </li><li> In Transfektionsgemisch tropfenweise zu den ekotropischen HEK 293-basierte Verpackungszellen. Vorsichtig die Platten wirbeln und sie zurück in die 5% CO 2, 37 ° C Inkubator zurück. Nach 24 Stunden ändern, um die Medien zu 10 ml frischem Wachstumsmedium vorgewärmten. </li><li> Ernten Sie den Überstand (mit Retrovirus) 48 Stunden nach der Transfektion mit einer Pipette und den Überstand in ein 50 ml Zentrifugenröhrchen mit. 10 ml neue warme Medien auf die Platte und wieder in den Inkubator. Halten der Überstand bei 4 ° C. Hinweis: Das Medium wird hinzugefügt sanft bis ter Seite des Bohrloches, um nicht die Zellschicht zu stören. </li><li> Ernten Sie die zweite Charge von Überstand 60 Stunden nach der Transfektion und bündeln es mit dem Überstand von 2.2.5. Zentrifuge bei 500 × g, 4 ° C für 7 min Zelldebris zu entfernen. </li><li> Den Überstand in ein neues 50 ml Zentrifugenröhrchen. Verwenden Retro C2C12-Zellen an diesem Punkt zu transduzieren durch Vorgehen 2.2.9 oder aliquoten zu Schritt und speichern Sie den Überstand bei -20 ° C für die zukünftige Verwendung. </li><li> Auftauen Retrovirus aus 2.2.7 bei Raumtemperatur, wenn ein gefrorener Lager hier verwendet wird. Hinweis: Vermeiden Sie mehrere Gefrier-Auftau-Zyklen. </li><li> Platte C2C12-Zellen am Tag der Transduktion 30 Ziel - 40% Konfluenz. <ol><li> Entsorgen Sie das Wachstumsmedium und waschen C2C12-Zellen mit 3 ml Raumtemperatur PBS. Entfernen Sie die PBS, 500 &amp; mgr; l 0,25% Trypsin-EDTA und Inkubation der Platte in einem 37 ° C, 5% CO 2 Inkubator 3 - 5 min. </li><li> Neutralisieren des Trypsins durch Zugabe von 3 ml Wachstums medium. Pipette 6 - 8 mal um die Zellen zu suspendieren. Zählen von Zellen mit einem Hämozytometer und fügen 8,0 x 10 5 C2C12 - Zellen auf eine 60 - mm - Platte. </li></ol></li><li> 0,5 ml Retrovirus ein 60-mm-Platte von Zellen in 3 ml Wachstumsmedium zu infizieren. Bringen Sie die Platte in den Inkubator. </li><li> Ersetzen durch 3 ml frisches Medium mit Selektionsantibiotikum ergänzt (Puromycin 1-3 ug / ml) 48 Stunden nach der Infektion. Ersetzen 3 ml Medium mit Antibiotikum jeden Tag für ~ 5 Tage, bis die untransduzierten C2C12 Kontrollkultur stirbt aus. </li></ol></li><li> Lentiviren Vorbereitung und C2C12 lentivirale Transduktion Hinweis: Führen Sie alle Lentiviren bezogene Verfahren in Biosafety Level 2 Schrank und biologische Sicherheitsrichtlinien befolgen. Flüssige Abfälle, das Virus enthalten, müssen vor der Entsorgung desinfiziert werden. <ol><li> Platte 4.0 x 10 6 293T - Zellen in einer 100 mm Platte (~ 80% Konfluenz am Tag der Transfektion) mit HEK 293 - Zellkulturmedium (DMEM high Glucose, ergänzt mit 10% fötalemRinderserum, 100 U / ml Penicillin, 100 ug / ml Streptomycin und 2 mM L-Glutamin) 24 h vor der Transfektion. </li><li> Wärmen Sie das Transfektionsreagenz auf Raumtemperatur. Mischen Sie die lentivirale Verpackungsvektoren (4 ug pMD2.G und 6 ug psPAX2) zusammen mit 8 ug lentiviralen Transfervektor (Celf1 shRNA oder verschlüsselt Kontrolle shRNA) in 1 ml Serum-freiem Zellkulturmedium. Vortex kurz. </li><li> In 36 ul vorgewärmte Transfektionsreagenz an die DNA / DMEM Rohr und gut mischen durch Wirbel. Inkubieren der Mischung bei Raumtemperatur für 30 min. </li><li> Entfernen Sie die 293T-Zellen aus dem Inkubator und zu der Platte die Transfektionsgemischs. Die Platte vorsichtig wirbeln und es Inkubator zurück zu 5% zurückkehren CO 2, 37 ° C. Nach 24 Stunden, ersetzen Sie das Transfektionsgemisch mit 10 ml frischem Kulturmedium. </li><li> Die überstehende Flüssigkeit enthält Lentiviren 48 Stunden nach der Transfektion und gibt sie in eine 50 ml Zentrifugenröhrchen. 10 ml neue warme Medien auf die Platte und senden Sie esInkubator. Lagern Sie den Überstand bei 4 ° C. </li><li> Sammeln Sie die zweite Charge von Überstand 60 Stunden nach der Transfektion und kombinieren es mit dem Überstand von 2.3.5. Zentrifuge kurz bei 500 · g, 4 ° C für 7 min. </li><li> Den Überstand in ein frisches 50 ml Zentrifugenröhrchen. Wenn frische Virus verwenden, fahren 2.3.9 zu treten. Für eine zukünftige Verwendung, Speicherung virushaltigen Überstand bei -20 ° C in Aliquots von 10 ml. </li><li> Auftauen das Virus aus 2.3.7 bei Raumtemperatur vor der Verwendung, wenn ein gefrorener Vorrat verwendet wird. Hinweis: Vermeiden Sie mehrere Gefrier-Auftau-Zyklen. </li><li> Platte C2C12-CUG200 Zellen (erhalten in Abschnitt 2.1), wie in 2.2.9 am gleichen Tag der Transduktion beschrieben. Hinweis: Ziel für 30 - 40% der Einmündung. </li><li> 0,5 ml Virus eine 60-mm-Platte von C2C12-CUG200 zu infizieren und die Platte in den Inkubator zurück. </li><li> Ersetzen Sie das Kulturmedium durch frisches Medium mit der Auswahl Antibiotikum ergänzt (Puromycin 1-3 ug / ml) 72 Stunden nach der Transduktion.Aktualisieren Kulturmedien jeden Tag mit dem Selektionsantibiotikum für ~ 5 Tage, bis alle untransduzierten C2C12-CUG200 Kontrollkultur stirbt aus. </li><li> Verwenden Western-Blot-Celf1 Knockdown in GFP-CUG200 transfizierten Zellen zu überprüfen. </li></ol></li></ol> 3. C2C12 Zelldifferenzierung <ol><li> Platte 2 x 10 6 Zellen in einer Vertiefung einer Platte mit 6 Vertiefungen in 2,5 ml Wachstumsmedium 24 h vor der Initiierung der Differenzierung. Hinweis: Die Beschichtungsdichte eingestellt werden kann, so dass eine vollständige Konfluenz in 24 h erreicht ist. <ol><li> Spülen konfluenten Zellen mit PBS und fügen Sie 2,5 ml serumarmen Differenzierungsmedium (Dulbeccos modifiziertem Eagle-Medium mit 2% Pferdeserum, 100 U / ml Penicillin, 100 ug / ml Streptomycin, 2 mM L-Glutamin und 1 uM Insulin) . Ersetzen Sie das Medium alle zwei Tage. Hinweis: Bewahren Sie Lager Insulin gefroren und fügen Sie es bei jedem Mediumwechsel zu den Medien. </li></ol></li><li> Während C2C12 Differenzierung, sammeln Proben für Quantitative RT-PCR in den folgenden Zeitpunkten (siehe Abschnitt 4): Tag0, 1, 2, 4 und 6. Verfahren die Kultur für Tag bei immunostaining 6 - 8 (siehe Abschnitt 5). </li></ol> 4. RNA Isolation und Quantitative RT-PCR <ol><li> Verwenden des Protokolls des Herstellers für die RNA-Isolierung. Nach der Isolierung resuspendieren RNA-Pellet in 30 ul RNase-freies Wasser und pipettieren nach oben und unten mehrmals. Hinweis: Die Proben Quantitative RT-PCR erfolgen kann oder kann bei -80 ° C gelagert werden. </li><li> Verwenden Sie einen RT - qPCR - Kit für die quantitative RT-PCR 10 und folgen Sie den Anweisungen des Herstellers. Bereiten Sie die Reaktionsmischung gemäß Tabelle 1. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Komponente </td><td> Volumen (ul) </td><td> Endkonzentration </td></tr><tr><td> 2x Reaktionspuffer </td><td> 10 </td><td> 1x </td&gt; </tr><tr><td> Vorwärtsprimer </td><td> 0,5 </td><td> 200 nM </td></tr><tr><td> Reverse-Primer </td><td> 0,5 </td><td> 200 nM </td></tr><tr><td> Sonde </td><td> 0,5 </td><td> 200 nM </td></tr><tr><td> Reverse Transkriptase und RNase Inhibitor </td><td> 0,1 </td><td> 0,25 U / ml </td></tr><tr><td> Vorlage </td><td> 1 </td><td> 100 ng Gesamt-RNA </td></tr><tr><td> RNase freies Wasser </td><td> 7.4 </td><td> N / A </td></tr><tr><td> insgesamt Mix </td><td> 20 </td><td></td></tr></tbody></table> Tabelle 1 : Quantitative RT-PCR Master Mix Herstellungs <ol start="3"><li> Führen Sie einen 20 &amp; mgr; l RT-qPCR - Reaktion unter Verwendung thermischen Profils Tabelle 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Reverse Transkriptionsschritt </td><td> 30 min, 48 ° C </td></tr><tr><td> DNA-Polymerase Aktivierung / Reverse-Transkriptase-Inaktivierung </td><td> 10 min, 95 ° C </td></tr><tr><td rowspan="2"> 40 Zyklen </td><td> 15 sec, 95 ° C </td></tr><tr><td> 1 min, 60 ° C </td></tr></tbody></table> Tabelle 2. Quantitative RT-PCR - Thermoprofil 5. Immunostaining <ol><li> Befestigen Sie die Monolayer-Kultur in 2,5 ml frisch 4% Paraformaldehyd / PBS bei Raumtemperatur für 10 Minuten vorbereitet. </li><li> Permeabilisieren die Proben in 2,5 ml 0,1% Triton X-100 / PBS für 30 min bei Raumtemperatur. </li><li> Übertragen Sie die Proben auf einer feuchten Kammer. Blockieren die Proben in 2,5 ml Blockierungspuffer (0,1% nichtionisches Tensid / PBS, 10% normales Ziegenserum) für 30 min bei 37 ° C. </li><li> Verwerfen Blockierungspuffer und anzuwenden 800 ul primärem Antikörper gegen Myosin schwere Kette in Blockierungspuffer verdünnt (1: 100). Inkubieren in einer befeuchteten Kammer über Nacht bei Raumtemperatur. </li><li> Waschen Sie die Platte 3 times (jeweils 10 Minuten) mit 2,5 ml Waschpuffer (0,1% Polysorbat 20 / PBS) am 2. Tag. </li><li> Entfernen der Waschpuffer und 800 ul gelten Sekundärantikörper (Ziege-anti-Maus-IgG, 1: 500 in PBS verdünnt). Inkubieren in einer befeuchteten Kammer bei Raumtemperatur für 1 Std. </li><li> Waschen zweimal (jeweils 10 Minuten) mit 2,5 ml Waschpuffer. </li><li> Inkubieren mit 800 &amp; mgr; l DAPI (1 ug / ml, in destilliertem deionisiertem H 2 O verdünnt) für 5 min. </li><li> Waschen mit 2,5 ml Waschpuffer wieder für 10 min. </li><li> Ändern Sie den Waschpuffer auf 2,5 ml PBS und mit einem Fluoreszenz-Mikroskop Bilder zu erfassen. </li></ol>

<ol>
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The authors have nothing to disclose.

C2C12 - Zelllinie wurde als Modell verwendet worden myogenesis 11-14 zu studieren. Diese Zellen behalten einen Fibroblasten-ähnliche Aussehen, proliferieren schnell in den Medien 20% fötales Rinderserum enthält , und unterscheiden sich leicht in Medien mit 2% Pferdeserum 15. Das schnelle Wachstum und die Differenzierung sind vorteilhafte Eigenschaften in einem myogenesis Zellmodell. Hier zeigen wir die Verwendung des Plasmids, retroviralen und lentiviralen Vektoren einzuführen cDNA, 3&#39;-UTR ...

Muskeldystrophie (DM) ist eine autosomal dominante Erkrankung , die mehrere Systeme betrifft, vor allem Herz- und Skelettmuskulatur 1. Es gibt zwei Subtypen dieser Krankheit, DM1 und DM2. DM1 ist häufiger und hat 2 eine schwere Manifestation als DM2. Die genetische Mutation DM1 zugrunde liegt , ist eine Erweiterung der CUG Triplett - Wiederholungen in der 3&#39; - untranslatierten Region liegt (UTR) von DM - Proteinkinase - Gen (DMPK) 3. Der CUG Wiederholungszahl in nicht betroffenen Individuen variiert von 5 bis 37. Im Gegensatz dazu erhöht sich auf mehr als 50, und manchmal bis zu Tausenden in DM1 Patienten 4. Als Ergebnis RNA-bindende Proteine, wie muscle-like 1 (MBNL1), CUGBP und Elav-like Familie 1 (Celf1) falsch reguliert sind. Aufgrund der Sequestrierung auf den CUG repeats erweitert, verliert seine Fähigkeit MBNL1 5 alternatives Spleißen zu regulieren. Celf1, andererseits wird 6,7 hochreguliert. Die Überexpression von Celf1 mit Muskelverlust verbundenund Schwäche, die zum Verlust nicht von MBNL1 Funktion zugeschrieben werden. Tiermodelle DM1 bedingte Veränderungen Simulieren, einschließlich DMPK 3&#39;-UTR CUG Expansion, Verlust von MBNL1 und Überexpression von Celf1, festgelegt wurden. Allerdings bietet die Modellierung DM1 in Myoblasten eine effiziente Alternative, vor allem für Sezieren DM1 bezogenen zellulären und molekularen Ereignisse. C2C12 Myoblasten - Zelllinie wurde zum ersten Mal von verletzten C3H Maus Muskel isoliert und weit verbreitete Muskeldifferenzierung 8,9 zu studieren. C2C12 Zellen vermehren sich rasch in fötalem Rinderserum (FBS) Medien -haltigen und leicht Differenzierung durchmachen, wenn FBS erschöpft ist. Doch dieses Myoblastendifferenzierung Modell stellt zwei Herausforderungen: C2C12-Zellen sind oft resistent gegen Gen-Transfektion / Virusübertragung; und leichte Variationen in die Handhabung von Zellen und Differenzierungsverfahren kann zu deutlichen Veränderungen in Myotube Bildung führen. Unser Labor routinemäßig verwendet C2C12 Myoblasten als acell - Modell und hat Protokolle entwickelt , die effektiv Gene durch Plasmidtransfektion 10, retrovirale Transduktion und lentivirale Transduktion in C2C12 - Zelllinie zu liefern. Im Video zeigen wir die optimierte Verfahren für die Transfektion / Transduktion C2C12-Zellen und die Differenzierung Konsistenz beibehalten in Modellen DM1 Myoblasten zu etablieren.

<table><tbody><tr><td>DMEM, high glucose</td><td>Life Technologies</td><td>11965-084</td><td>for culture medium</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>for culture medium</td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100x)</td><td>Life Technologies</td><td>10378-016</td><td>for culture medium</td></tr><tr><td>Insulin from bovine pancreas</td><td>Sigma Aldrich</td><td>I6634-100MG</td><td>for differentiation medium</td></tr><tr><td>equine serum</td><td>Atlanta Biologicals</td><td>S12150</td><td>for differentiation medium</td></tr><tr><td>FuGENE&amp;nbsp;HD Transfection Reagent</td><td>Promega</td><td>E2311&amp;nbsp;</td><td>for transfection</td></tr><tr><td>G418 sulfate&amp;nbsp;</td><td>Gold Biotechnology&amp;nbsp;</td><td>G-418-10</td><td>for drug resistant selection</td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma Aldrich</td><td>sc-108071</td><td>for drug resistant selection</td></tr><tr><td>NuPAGE Novex 4 - 12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td><td>Life Technologies</td><td>NP0323BOX</td><td>for western blot</td></tr><tr><td>NuPAGE Transfer Buffer (20x)</td><td>Life Technologies</td><td>NP00061</td><td>for western blot</td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Life Technologies</td><td>NP0002</td><td>for western blot</td></tr><tr><td>Amersham Protran Supported 0.2 NC, 300 mm x 4 m</td><td>GE healthcare life science</td><td>10600015</td><td>for western blot</td></tr><tr><td>MF 20</td><td>Developmental Hybridoma Bank</td><td>MF 20</td><td>primary Ab for immunostaining</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate</td><td>Thermo Fisher Scientific</td><td>T-862</td><td>secondary Ab for immunostaining</td></tr><tr><td>One step qRT-PCR MasterMix</td><td>AnaSpec</td><td>05-QPRT-032X</td><td>for qRT-PCR</td></tr><tr><td>TriPure Isolation Reagent</td><td>Roche</td><td>11667165001</td><td>for RNA isolation</td></tr><tr><td>CUG-BP1 Antibody (3B1)</td><td>santa cruz</td><td>sc-20003</td><td>primary Ab western blot</td></tr><tr><td>Actin Antibody</td><td>santa cruz</td><td>sc-1615</td><td>goat polyclonal IgG for loading control</td></tr><tr><td>293T Ecopack</td><td>Clontech</td><td>631507</td><td>cells for retrovirus preparation</td></tr><tr><td>pMSCV-puro</td><td>Clontech</td><td>634401</td><td>empty retroviral vector for retrovirus preparation</td></tr><tr><td>pMSCV-Celf1Flag-puro</td><td>house-constructed</td><td>not available</td><td>retroviral vector encoding Celf1Flag, used in retrovirus preparation</td></tr><tr><td>psPAX2</td><td>gift from Didier Trono</td><td>not available</td><td>for lentivirus preparation</td></tr><tr><td>pMD2.G</td><td>gift from Didier Trono</td><td>not available</td><td>for lentivirus preparation</td></tr><tr><td>GFP-CUG5</td><td>gift from M.S. Mahadevan</td><td>not available</td><td>details in reference 10&amp;nbsp;</td></tr><tr><td>GFP- CUG200</td><td>gift from M.S. Mahadevan</td><td>not available</td><td>details in reference 10&amp;nbsp;</td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>X100</td><td>for immunostaining</td></tr><tr><td>paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>for immunostaining</td></tr><tr><td>TWEEN&amp;nbsp;20</td><td>Sigma Aldrich</td><td>P9416</td><td>for immunostaining</td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td>for immunostaining</td></tr></tbody></table>

modeling myotonic dystrophy 1 in c2c12 myoblast cells

Muskeldystrophie 1 (DM1) ist eine häufige Form der Muskeldystrophie. Obwohl mehrere Tiermodelle haben für DM1, Myoblasten Zellmodelle sind nach wie vor wichtig, weil sie eine effiziente zelluläre Alternative für die Untersuchung zellulärer und molekularer Ereignisse bieten etabliert. Obwohl in großem Umfang Zellen C2C12 Myoblasten verwendet myogenesis, Resistenz gegen Gen-Transfektion oder virale Transduktion zu studieren, behindert die Forschung in C2C12-Zellen. Hier beschreiben wir ein optimiertes Protokoll, das die tägliche Wartung umfasst, Transfektion und Transduktion Verfahren Gene in C2C12 Myoblasten und die Induktion von myocyte Differenzierung einzuführen. Gemeinsam ermöglichen diese Verfahren am besten Transfektion / Transduktionseffizienzen sowie konsequente Differenzierung Ergebnisse. Das Protokoll bei der Einrichtung DM1 Myoblasten Zellmodelle beschrieben würde die Studie von Muskeldystrophie zugute kommen, sowie andere Muskelerkrankungen.

C2C12-Zellen wurden mit GFP-CUG5 oder GFP-CUG200 transfiziert. Nach Medikamentenresistenz Selektion wurden stabile Pools hergestellt, das durch die GFP - Expression (Abbildung 1A) sichtbar gemacht werden kann. Myotube Bildung in den differenzierten Myoblasten wurde von Myosin schwere Kette Immunofärbung 10 (1B) detektiert. Die Quantifizierung der Myotube Bildung zeigte , dass Fusions Indizes von 35,4 ± 4,1% auf 2,6 ± 1,1% und Myotube Bereic...

Watch this Scientific Journal Video about Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells at JoVE.com

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

In diesem Protokoll stellen wir die Verfahren in Muskeldystrophie 1 Myoblasten Modelle Gründung, einschließlich optimierte C2C12 Zell Wartung, Gen-Transfektion / Transduktion und Myozyten-Differenzierung.

Modellierung Muskeldystrophie 1 in C2C12 Myoblastenzellen

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

modeling-myotonic-dystrophy-1-in-c2c12-myoblast-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.2K Aufrufe.  University of Houston.  In diesem Protokoll stellen wir die Verfahren in Muskeldystrophie 1 Myoblasten Modelle Gründung, einschließlich optimierte C2C12 Zell Wartung, Gen-Transfektion / Transduktion und Myozyten-Differenzierung. 

Serie: Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineering Skeletal Muscle Gewebe von Murine Myoblast Progenitor Cells und Anwendung von Elektrostimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Forschung

Biotechnik

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

In vitro Differenzierung von Mature Muskelfasern für Live - Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Entwicklungsbiologie

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Ein Functional Motor Unit in der Kulturschale: Co-Kultur von Spinal Cord Explantate und Muskelzellen

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Neurowissenschaften

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Molecular Imaging Transplantierte Muscle Progenitor Zielzellen

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Medizin

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Stabiler Knockdown von Genen, die extrazelluläre Matrixproteine in der C2C12 Myoblast-Zelllinie mit Small-Hairpin (sh)RNA kodieren

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

Genetik

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.

Direkte Umprogrammierung menschlicher Fibroblasten in Myoblasten zur Untersuchung von Therapien für neuromuskuläre Störungen

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

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolierung und Differenzierung von primären Myoblasten von Mausskelettmuskelexplants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolierung und Immortalisierung von Patienten stammende Zelllinien von Muskelbiopsie for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Charcot-Marie-Zahn Krankheit In Vitro Modellierung durch Transfecting Maus primären Motoneurone

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

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).

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