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 coltura cellulare <ol><li> Mantenere C2C12 mioblasti di topo in un piatto di 100 mm di mezzo di crescita (media di Dulbecco modificato Eagle (DMEM)) integrato con il 20% di siero fetale bovino, 100 U / ml di penicillina, 100 ug / ml di streptomicina, e 2 mm L-glutammina. Consentono alle cellule C2C12 diversi passaggi a diventare circa il 50 - 60% confluenti. </li><li> Eliminare il mezzo di crescita e lavare le cellule C2C12 con soluzione fisiologica 3 ml di temperatura ambiente tamponata con fosfato (PBS). Rimuovere il PBS e aggiungere 500 microlitri 0,25% tripsina-EDTA per staccare le cellule. Posizionare la piastra a 37 ° C, 5% CO 2 incubatore 3 - 5 min. </li><li> Neutralizzare la tripsina con l&#39;aggiunta di 3 ml di terreno di coltura. Pipettare 6 - 8 volte per sospendere le cellule. Aggiungere 1 ml di cellule sospese in un piatto di 100 mm e incubare a 37 ° C, 5% CO 2. </li></ol> 2. C2C12 cellule Trasfezione / trasduzione e Selezione <ol><li> C2C12 plasmide trasfezione <ol><li> 24 ore prima della trasfezione, pritardate 7,0 x 10 5 cellule C2C12 in un pozzetto di una piastra sei pozzetti per ogni trasfezione. Nota: Questo porta a 50 - 60% di confluenza al momento della transfezione. </li><li> Lasciare che il reagente di trasfezione raggiunga la temperatura ambiente prima dell&#39;uso. </li><li> Aggiungere 2 mg di DNA plasmide (GFP-CUG5 o GFP-CUG200) in 200 ml di pre-riscaldato mezzo privo di siero e vortex brevemente. Aggiungere 6 ml di reagente di trasfezione al mezzo e miscelare immediatamente per 30 secondi usando un vortice. Incubare la miscela per 30 minuti a temperatura ambiente. </li><li> Cambiare il supporto di cellule C2C12 a 2,5 ml terreni di coltura privo di siero. Aggiungere la miscela di trasfezione di cellule dopo incubazione. Riportare le piastre per un 5% di CO 2, 37 ° C incubatore. </li><li> Mantenere le cellule in miscela di trasfezione / terreni di crescita senza siero per 4 ore o fino a notte. Cambiare i media indietro ai media di crescita il giorno successivo o quando citotossicità è evidente. Nota: La citotossicità è valutata mediante ispezione microscopica del ceLLS. Osservare i cambiamenti nella morfologia cellulare e il distacco delle cellule. Fino a quando non vi è alcuna citotossicità palese, l&#39;incubazione durante la notte in terreni di crescita privo di siero migliora trasfezione. </li><li> Selezionare le celle 48 ore dopo la trasfezione con l&#39;aggiunta di 1,2-1,6 mg / ml G418 per ~ 10 giorni fino a quando la cultura del controllo (transfettate senza i plasmidi) non ha cellule vive. Modificare i media ogni due giorni con mezzi di crescita integrato con G418. </li><li> Mantenere le cellule resistenti G418 in mezzo di crescita e 5% di CO 2 a 37 ° C per esperimenti successivi. </li></ol></li><li> preparazione Retrovirus e C2C12 retrovirale trasduzione <ol><li> Preparare HEK 293 terreno di coltura DMEM completandolo glucosio con il 10% di siero fetale bovino, 100 U / ml di penicillina, 100 ug / ml streptomicina, e 2 mM L-glutammina. <ol><li> Circa 24 ore prima della trasfezione, piatto 4,0 x 10 cellule di packaging HEK 293 basati su 6 ecotropic in un 100 mm Piastra (80% di confluenza a trasfezione) cmezzo di coltura cellulare ontaining. </li></ol></li><li> Lasciare che il reagente di trasfezione raggiunga la temperatura ambiente prima dell&#39;uso. </li><li> Mescolare 15 mcg plasmide DNA (pMSCV-Puro o pMSCV-Celf1Flag-Puro) in 1 ml di terreno di coltura privo di siero. brevemente Vortex. Aggiungere 30 ml di reagente di trasfezione dal punto 2.2.2 in / tubo di terreno di coltura privo di siero del DNA, e mescolare bene con vortex. Incubare la miscela a temperatura ambiente per 30 min. </li><li> Aggiungere trasfezione miscela goccia a goccia per le cellule di confezionamento ecotropic HEK 293-based. Mescolare delicatamente i piatti e li tornare al 5% di CO 2, 37 ° C incubatore. Dopo 24 ore, cambiare i mezzi di comunicazione per mezzo di crescita fresco 10 ml di pre-riscaldato. </li><li> Raccogliere il surnatante (contenente retrovirus) 48 ore dopo la trasfezione con una pipetta e trasferire il surnatante in una provetta da centrifuga da 50 ml. Aggiungere 10 ml di nuovi media caldi alla piastra e restituirlo al incubatrice. Mantenere il surnatante a 4 ° C. Nota: Il supporto viene aggiunto delicatamente per tegli lato del pozzo in modo da non disturbare il monostrato cellulare. </li><li> Raccolto il secondo lotto di surnatante 60 ore dopo la trasfezione e piscina con il surnatante da 2.2.5. Centrifugare a 500 xg, a 4 ° C per 7 minuti per rimuovere i detriti cellulari. </li><li> Trasferire il surnatante in un nuovo tubo da 50 ml centrifuga. Utilizzare retrovirus di trasdurre cellule C2C12 a questo punto procedendo al punto 2.2.9 o aliquotare e conservare il surnatante a -20 ° C per un uso futuro. </li><li> Scongelare il retrovirus da 2.2.7 a temperatura ambiente, se un titolo congelato è qui utilizzato. Nota: Evitare ripetuti cicli di congelamento-scongelamento. </li><li> Piastra cellule C2C12 lo stesso giorno di trasduzione puntando 30 - 40% di confluenza. <ol><li> Eliminare il mezzo di crescita e lavare le cellule C2C12 con temperatura ambiente 3 ml di PBS. Rimuovere la PBS, aggiungere 500 microlitri 0,25% tripsina-EDTA e incubare la piastra a 37 ° C, 5% CO 2 incubatore 3 - 5 min. </li><li> Neutralizzare la tripsina con l&#39;aggiunta di 3 ml med crescitaIUM. Pipettare 6 - 8 volte per sospendere le cellule. Contare le cellule utilizzando un emocitometro e aggiungere 8,0 x 10 5 cellule C2C12 ad una piastra 60 mm. </li></ol></li><li> Aggiungere 0,5 ml retrovirus di infettare un piatto 60 mm di cellule in 3 ml di mezzi di crescita. Riportare la piastra per l&#39;incubatore. </li><li> Sostituire con 3 supporti ml freschi integrato con selezione antibiotica (puromicina 1 - 3 mg / ml) 48 ore dopo l&#39;infezione. Sostituire 3 ml di mezzo con antibiotici ogni giorno per ~ 5 giorni fino a quando la cultura del controllo C2C12 non trasdotte spegne. </li></ol></li><li> preparazione lentivirus e C2C12 lentiviral trasduzione Nota: eseguire tutte le procedure lentivirus legati a livello di biosicurezza 2 mobile e seguire le linee guida di sicurezza biologica. I rifiuti liquidi contenenti il ​​virus devono essere disinfettati prima dello smaltimento. <ol><li> Piatto 4,0 x 10 6 cellule 293T in un piatto 100 mm (~ 80% di confluenza al giorno di trasfezione) con HEK 293 terreno di coltura cellulare (DMEM alti livelli di glucosio, integrato con il 10% del fetosiero bovino, 100 U / ml di penicillina, 100 ug / ml streptomicina, e 2 mM L-glutammina) 24 ore prima della transfezione. </li><li> Riscaldare il reagente di trasfezione a temperatura ambiente. Mescolare i vettori lentivirali di confezionamento (4 mg pMD2.G e 6 mcg psPAX2) insieme con 8 mg di trasferimento vettori lentivirali (Celf1 shRNA o strapazzate controllo shRNA) in 1 ml di siero mezzo di coltura cellulare gratuito. brevemente Vortex. </li><li> Aggiungere 36 ml di pre-riscaldato reagente di trasfezione al tubo DNA / DMEM e mescolare bene dal vortice. Incubare la miscela a temperatura ambiente per 30 min. </li><li> Rimuovere le cellule 293T dal termostato e aggiungere la miscela di trasfezione alla piastra. Agitare delicatamente la piastra e tornare di nuovo al 5% di CO 2, 37 ° C incubatore. Dopo 24 ore, sostituire la miscela di trasfezione con 10 ml di terreno di coltura fresco. </li><li> Raccogliere il surnatante contenente lentivirus 48 ore dopo la trasfezione e trasferirlo in una provetta da centrifuga da 50 ml. Aggiungere 10 ml nuovi media caldi alla piastra e restituirloper incubatrice. Conservare il surnatante a 4 ° C. </li><li> Raccogliere il secondo lotto di surnatante 60 ore dopo la trasfezione e combinarlo con il surnatante da 2.3.5. brevemente Centrifugare a 500 xg, a 4 ° C per 7 minuti. </li><li> Trasferire il surnatante in un tubo da 50 ml centrifuga fresca. Se si utilizza il virus fresco, procedere al punto 2.3.9. Per l&#39;uso futuro, negozio di virus contenenti surnatante a -20 ° C in aliquote di 10 ml. </li><li> Scongelare il virus da 2.3.7 a temperatura ambiente prima dell&#39;uso, se si utilizza un magazzino congelato. Nota: Evitare ripetuti cicli di congelamento-scongelamento. </li><li> cellule piastra C2C12-CUG200 (ottenuti nella sezione 2.1), come descritto nella 2.2.9 lo stesso giorno di trasduzione. Nota: Obiettivo per 30 - 40% di confluenza. </li><li> Aggiungere 0,5 ml di virus di infettare una piastra 60 mm C2C12-CUG200 e riportare la piastra per l&#39;incubatore. </li><li> Sostituire il terreno di coltura con mezzi freschi integrati con l&#39;antibiotico selezione (puromicina 1 - 3 mg / ml) 72 ore dopo la trasduzione.Aggiornare mezzi di coltura di tutti i giorni con l&#39;antibiotico selezione di ~ 5 giorni fino a quando tutta la cultura di controllo C2C12-CUG200 non trasdotte spegne. </li><li> Utilizzare Western Blot per verificare atterramento Celf1 in cellule trasfettate GFP-CUG200. </li></ol></li></ol> 3. C2C12 cellule Differenziazione <ol><li> Piastra 2 x 10 6 cellule in un pozzetto di una piastra da 6 pozzetti in 2,5 ml terreni di crescita 24 h prima apertura di differenziazione. Nota: la densità di placcatura può essere regolata in modo che la piena confluenza viene raggiunto in 24 ore. <ol><li> Lavare le cellule confluenti con PBS e aggiungere 2,5 ml di terreno di differenziamento basso siero (modificato mezzo di Eagle integrato con 2% di siero equino, 100 U / ml penicillina Dulbecco, 100 ug / ml di streptomicina, 2 mM L-glutammina e 1 pM insulina) . Sostituire la media ogni due giorni. Nota: Mantenere magazzino insulina congelati e aggiungerlo ai media ad ogni cambio medio. </li></ol></li><li> Durante la differenziazione C2C12, raccogliere campioni per quanquantita- RT-PCR (vedi sezione 4) presso i seguenti punti di tempo: Day0, 1, 2, 4 e 6. Processo per la cultura immunocolorazione al giorno 6-8 (vedi sezione 5). </li></ol> 4. RNA Isolamento e RT-PCR quantitativa <ol><li> Utilizzare il protocollo del produttore per l&#39;isolamento di RNA. Dopo l&#39;isolamento, risospendere il pellet di RNA in 30 acqua priva di RNasi ml e pipettare su e giù parecchie volte. Nota: I campioni possono procedere alla RT-PCR quantitativa o possono essere conservati a -80 ° C. </li><li> Utilizzare un kit RT qPCR per RT-PCR quantitativa 10 e seguire le istruzioni del produttore. Preparare la miscela di reazione in base alla tabella 1. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Componente </td><td> Volume (ml) </td><td> Concentrazione finale </td></tr><tr><td> tampone di reazione 2x </td><td> 10 </td><td> 1x </td&gt; </tr><tr><td> Primer forward </td><td> 0.5 </td><td> 200 nm </td></tr><tr><td> primer reverse </td><td> 0.5 </td><td> 200 nm </td></tr><tr><td> Sonda </td><td> 0.5 </td><td> 200 nm </td></tr><tr><td> Trascrittasi inversa e RNase Inhibitor </td><td> 0.1 </td><td> 0,25 U / ml </td></tr><tr><td> Modello </td><td> 1 </td><td> 100 ng RNA totale </td></tr><tr><td> acqua libera RNase </td><td> 7.4 </td><td> N / A </td></tr><tr><td> Mix totale </td><td> 20 </td><td></td></tr></tbody></table> Tabella 1. RT-PCR Master Mix Preparazione <ol start="3"><li> Eseguire un 20 microlitri reazione RT-qPCR con profilo termico Tabella 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> fase di trascrizione inversa </td><td> 30 min, 48 ° C </td></tr><tr><td> polimero DNAAttivazione ase / trascrittasi inversa inattivazione </td><td> 10 min, 95 ° C </td></tr><tr><td rowspan="2"> 40 cicli </td><td> 15 sec, 95 ° C </td></tr><tr><td> 1 min, 60 ° C </td></tr></tbody></table> Tabella profilo termico 2. RT-PCR quantitativa 5. immunocolorazione <ol><li> Fissare la coltura monostrato in 2.5 ml preparato fresco 4% paraformaldeide / PBS a temperatura ambiente per 10 min. </li><li> Permeabilize i campioni in 2.5 ml 0,1% Triton X-100 / PBS per 30 minuti a temperatura ambiente. </li><li> Trasferire i campioni ad una camera umidificata. Bloccare i campioni in 2,5 ml di tampone di bloccaggio (tensioattivo non ionico 0,1% / PBS, 10% siero di capra normale) per 30 minuti a 37 ° C. </li><li> Scartare tampone bloccante e applicare 800 microlitri anticorpo primario contro catena pesante della miosina diluito in tampone di bloccaggio (1: 100). Incubare in una camera umidificata notte a temperatura ambiente. </li><li> Lavare la piastra 3 times (10 min ciascuna) con 2,5 ml di tampone di lavaggio (0,1% polisorbato 20 / PBS) il giorno 2. </li><li> Rimuovere il tampone di lavaggio e applicare 800 microlitri anticorpo secondario (goat anti-topo IgG, 1: 500 diluito in PBS). Incubare in una camera umidificata a temperatura ambiente per 1 ora. </li><li> Lavare due volte (10 minuti ciascuno) con tampone di 2,5 ml di lavaggio. </li><li> Incubare con 800 microlitri DAPI (1 ug / ml, diluito in acqua distillata deionizzata H 2 O) per 5 min. </li><li> Lavare con 2,5 ml di tampone di lavaggio ancora per 10 min. </li><li> Modificare il buffer di lavaggio a 2,5 ml di PBS e utilizzare un microscopio a fluorescenza per catturare le immagini. </li></ol>

<ol>
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The authors have nothing to disclose.

Linea cellulare C2C12 è stata utilizzata come modello per lo studio miogenesi 11-14. Queste cellule mantengono uno sguardo fibroblasti-like, proliferano rapidamente in supporti contenenti il 20% di siero fetale bovino e prontamente si differenziano nel supporto contenente 2% di siero equino 15. La rapida crescita e la differenziazione sono caratteristiche vantaggiose in un modello cellulare miogenesi. Qui, dimostriamo l&#39;uso di plasmide, retrovirale, e vettori lentivirali per introdurre cDNA, 3...

La distrofia miotonica (DM) è una malattia autosomica dominante che colpisce di più sistemi, i muscoli più considerevolmente cardiaco e scheletrico 1. Ci sono due sottotipi di questa malattia, DM1 e DM2. DM1 è più comune e ha una manifestazione più grave di DM2 2. La mutazione genetica sottostante DM1 è una espansione della tripletta ripetizioni CUG situato nella regione 3 &#39;non tradotta (UTR) del DM gene della proteina chinasi (DMPK) 3. Il numero CUG repeat in individui sani varia da 5 a 37. Al contrario, aumenta di più di 50, e, a volte fino a migliaia di pazienti DM1 4. Di conseguenza, RNA-binding proteins, come muscleblind-simile 1 (MBNL1), CUGBP, e Elav-come la famiglia 1 (Celf1), sono misregulated. A causa del sequestro sulle ripetizioni CUG espanse, MBNL1 perde la sua capacità di regolare splicing alternativo 5. Celf1, invece, viene up-regolata 6,7. Sovraespressione di Celf1 è associata a perdita di massa muscolaree la debolezza, che non sono attribuiti alla perdita di funzione MBNL1. Modelli animali che simulano le alterazioni DM1, comprese l&#39;DMPK 3&#39;-UTR espansione CUG, perdita di MBNL1, e sovraespressione di Celf1, sono state stabilite. Tuttavia, la modellazione DM1 nei mioblasti offre una valida alternativa, soprattutto per sezionare gli eventi cellulari e molecolari DM1-correlati. Linea di cellule mioblasti C2C12 è stato isolato dal muscolo C3H topo feriti e ampiamente usato per studiare miogenico 8,9 differenziazione. C2C12 rapidamente proliferano in siero fetale bovino (FBS) -contenenti media e facilmente subiscono differenziazione quando FBS è esaurito. Tuttavia, utilizzando questo modello di differenziazione dei mioblasti presenta due sfide: C2C12 sono spesso resistenti al gene trasfezione / trasduzione virale; e lievi variazioni nella gestione delle cellule e differenziazione procedura può portare a notevoli cambiamenti in formazione myotube. Il nostro laboratorio utilizza abitualmente mioblasti C2C12 come ACmodello ell e ha sviluppato protocolli in grado di fornire in modo efficace i geni di plasmide trasfezione, trasduzione retrovirale, e lentivirale trasduzione nella linea cellulare C2C12 10. Nel video, dimostriamo le procedure ottimizzate per transfecting / trasduzione C2C12 cellule e mantenere la coerenza di differenziazione nella creazione di modelli di mioblasti DM1.

<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

La distrofia miotonica 1 (DM1) è una comune forma di distrofia muscolare. Anche se diversi modelli animali sono stati stabiliti per DM1, modelli cellulari mioblasti sono ancora importanti perché offrono una valida alternativa cellulare per studiare gli eventi cellulari e molecolari. Anche se le cellule mioblasti C2C12 sono stati ampiamente utilizzati per studiare miogenesi, resistenza alla trasfezione del gene, o trasduzione virale, ostacola la ricerca nelle cellule C2C12. Qui, descriviamo un protocollo ottimizzato che include le procedure di manutenzione quotidiana, trasfezione e trasduzione per introdurre geni in mioblasti C2C12 e l&#39;induzione della differenziazione dei miociti. Collettivamente, queste procedure consentono migliori efficienze di trasfezione / trasduzione, come pure risultati differenziazione coerenti. Il protocollo descritto nello stabilire modelli cellulari mioblasti DM1 beneficerebbero lo studio della distrofia miotonica, nonché altre malattie muscolari.

C2C12 sono state trasfettate con GFP-CUG5 o GFP-CUG200. Dopo la selezione farmaco-resistenza, sono stati stabiliti piscine stabili, che può essere visualizzato mediante espressione GFP (Figura 1A). Formazione myotube nei mioblasti differenziati è stato rilevato dal catena pesante della miosina immunocolorazione 10 (Figura 1B). La quantificazione di myotube formazione ha dimostrato che gli indici di fusione sono stati diminuiti da 35,4 ± 4,1...

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 questo protocollo, vi presentiamo le procedure nella creazione di modelli miotonici mioblasti distrofia 1, tra cui ottimizzato manutenzione C2C12 cellule, geni trasfezione / trasduzione, e la differenziazione dei miociti.

Modellazione Distrofia miotonica 1 nelle cellule dei mioblasti C2C12

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

Research

JoVE Journal

Medicine

15.3K Views.  University of Houston.  In questo protocollo, vi presentiamo le procedure nella creazione di modelli miotonici mioblasti distrofia 1, tra cui ottimizzato manutenzione C2C12 cellule, geni trasfezione / trasduzione, e la differenziazione dei miociti. 

Video: 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.

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

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 ...

Ricerca

Bioingegneria

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.

La differenziazione in vitro di miofibre mature per 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 ...

Biologia dello sviluppo

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.

Un gruppo motore funzionale nella piastra di coltura: Co-coltura degli espianti midollo spinale e le cellule muscolari

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

Neuroscienze

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.

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

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

Medicina

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.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins nella linea cell Myoblast C2C12 Utilizzando 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 ...

Genetica

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.

Riprogrammazione diretta dei fibroblasti umani in mioblasti per indagare le terapie per i disturbi neuromuscolari

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.

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

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.

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia 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.

Malattia di Charcot-Marie-Tooth In Vitro di modellazione di trasfezione motoneuroni primari del Mouse

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).

Isolamento e cultura della miofibra singola da un modello Murine della distrofia muscolare di Emery-Dreifuss nei primi sviluppi post-Natal

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

Modellazione Distrofia miotonica 1 nelle cellule dei mioblasti C2C12

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

La differenziazione in vitro di miofibre mature per Live Imaging

Un gruppo motore funzionale nella piastra di coltura: Co-coltura degli espianti midollo spinale e le cellule muscolari

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins nella linea cell Myoblast C2C12 Utilizzando Small-Hairpin (sh)RNA

Riprogrammazione diretta dei fibroblasti umani in mioblasti per indagare le terapie per i disturbi neuromuscolari

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

Isolamento e Immortalizzazione di paziente derivati da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

Malattia di Charcot-Marie-Tooth In Vitro di modellazione di trasfezione motoneuroni primari del Mouse

Isolamento e cultura della miofibra singola da un modello Murine della distrofia muscolare di Emery-Dreifuss nei primi sviluppi post-Natal

Modellazione Distrofia miotonica 1 nelle cellule dei mioblasti C2C12

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

La differenziazione in vitro di miofibre mature per Live Imaging

Un gruppo motore funzionale nella piastra di coltura: Co-coltura degli espianti midollo spinale e le cellule muscolari

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins nella linea cell Myoblast C2C12 Utilizzando Small-Hairpin (sh)RNA

Riprogrammazione diretta dei fibroblasti umani in mioblasti per indagare le terapie per i disturbi neuromuscolari

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

Malattia di Charcot-Marie-Tooth In Vitro di modellazione di trasfezione motoneuroni primari del Mouse

Isolamento e cultura della miofibra singola da un modello Murine della distrofia muscolare di Emery-Dreifuss nei primi sviluppi post-Natal

Isolamento e Immortalizzazione di paziente derivati da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling