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.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, high glucose</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">11965-084</td>
			<td style="width:325px;">for culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premium</td>
			<td style="width:112px;">Atlanta Biologicals</td>
			<td style="width:151px;">S11150</td>
			<td>for culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin-Glutamine (100X)</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">10378-016</td>
			<td>for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin from bovine pancreas</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">I6634-100MG</td>
			<td>for differentiation medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">equine serum</td>
			<td style="width:112px;">Atlanta Biologicals</td>
			<td style="width:151px;">S12150</td>
			<td>for differentiation medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FuGENE&#160;HD&#160;Transfection Reagent</td>
			<td style="width:112px;">Promega</td>
			<td style="width:151px;">E2311&#160;</td>
			<td>for transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">G418 sulfate&#160;</td>
			<td style="width:112px;">Gold Biotechnology&#160;</td>
			<td style="width:151px;">G-418-10</td>
			<td>for drug resistant selection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">sc-108071</td>
			<td>for drug resistant selection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">NP0323BOX</td>
			<td>for western blot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE Transfer Buffer (20X)</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">NP00061</td>
			<td>for western blot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE MES SDS Running Buffer (20X)</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">NP0002</td>
			<td>for western blot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amersham Protran Supported 0.2 NC, 300mmx4m</td>
			<td style="width:112px;">GE healthcare life science</td>
			<td style="width:151px;">10600015</td>
			<td>for western blot</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MF 20</td>
			<td style="width:112px;">Developmental Hybridoma Bank</td>
			<td style="width:151px;">MF 20</td>
			<td>primary Ab for immunostaining</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate</td>
			<td style="width:112px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">T-862</td>
			<td>secondary Ab for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">One step qRT-PCR MasterMix</td>
			<td style="width:112px;">AnaSpec</td>
			<td style="width:151px;">05-QPRT-032X</td>
			<td>for qRT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TriPure Isolation Reagent</td>
			<td style="width:112px;">Roche</td>
			<td style="width:151px;">11667165001</td>
			<td>for RNA isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CUG-BP1 Antibody (3B1)</td>
			<td style="width:112px;">santa cruz</td>
			<td style="width:151px;">sc-20003</td>
			<td>primary Ab western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Actin Antibody</td>
			<td style="width:112px;">santa cruz</td>
			<td style="width:151px;">sc-1615</td>
			<td>goat polyclonal IgG for loading control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">293T Ecopack</td>
			<td style="width:112px;">Clontech</td>
			<td style="width:151px;">631507</td>
			<td>cells for retrovirus preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pMSCV-puro</td>
			<td style="width:112px;">Clontech</td>
			<td style="width:151px;">634401</td>
			<td>empty retroviral vector for retrovirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pMSCV-Celf1Flag-puro</td>
			<td style="width:112px;">house-constructed</td>
			<td style="width:151px;">not available</td>
			<td>retroviral vector encoding Celf1Flag, used in retrovirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">psPAX2</td>
			<td style="width:112px;">gift from Didier Trono</td>
			<td style="width:151px;">not available</td>
			<td>for lentivirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pMD2.G</td>
			<td style="width:112px;">gift from Didier Trono</td>
			<td style="width:151px;">not available</td>
			<td>for lentivirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GFP-CUG5</td>
			<td style="width:112px;">gift from M.S. Mahadevan</td>
			<td style="width:151px;">not available</td>
			<td>details in reference 10&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GFP- CUG200</td>
			<td style="width:112px;">gift from M.S. Mahadevan</td>
			<td style="width:151px;">not available</td>
			<td>details in reference 10&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton&#160;X-100</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">X100</td>
			<td>for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">P6148</td>
			<td>for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TWEEN&#160;20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">P9416</td>
			<td>for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">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

15.1K 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

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Modellazione emorragia intracerebrale in Mouse: iniezione di sangue autologo o collagenasi batterica

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Ricerca

Medicina

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modellazione di Carcinoma renale metastatico spontanea (MRCC) in topi dopo nefrectomia

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Corsa di modellazione in Mouse: Coagulazione permanente dell&#39;arteria cerebrale media distale

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Generazione di pluripotenti indotte cellule staminali da distrofia muscolare di pazienti: Efficient Riprogrammazione senza integrazione di origine urinaria Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

Modellazione chemioterapia leucemia resistente<em&gt; In Vitro</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype&#160;is&#160;more similar to human DMD patients, was&#160;used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Exon skipping is currently a&#160;most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

Multi-esone skipping Utilizzando oligonucleotidi antisenso Cocktail nella distrofia muscolare Canine X-linked

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

Valutazione dell'inclusione dell'esone indotta da impiombare commutazione oligonucleotidi antisenso in fibroblasti del paziente di SMA

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...