Name: modeling myotonic dystrophy 1 in c2c12 myoblast cells
Uploaded: 2016-07-29T14:00:00
Description: Watch this Scientific Journal Video about Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells at JoVE.com

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). Ri...

<ol>
	<li>Harper, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Myotonic dystrophy. 3rd edn. , (2001).</li><li>Timchenko, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+muscle+atrophy+in+myotonic+dystrophies.">Molecular mechanisms of muscle atrophy in myotonic dystrophies.</a> Int J Biochem Cell Biol. 45 (10), 2280-2287 (2013).</li><li>Brook, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+myotonic+dystrophy:+expansion+of+a+trinucleotide+(CTG)+repeat+at+the+3'+end+of+a+transcript+encoding+a+protein+kinase+family+member.">Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member.</a> Cell. 68 (4), 799-808 (1992).</li><li>Chau, A., Kalsotra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+insights+into+the+pathology+of+and+therapeutic+strategies+for+DM1:+Back+to+the+basics.">Developmental insights into the pathology of and therapeutic strategies for DM1: Back to the basics.</a> Dev Dyn. 244 (3), 377-390 (2015).</li><li>Ho, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscleblind+proteins+regulate+alternative+splicing.">Muscleblind proteins regulate alternative splicing.</a> EMBO J. 23 (15), 3103-3112 (2004).</li><li>Kuyumcu-Martinez, N. M., Wang, G. S., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+steady-state+levels+of+CUGBP1+in+myotonic+dystrophy+1+are+due+to+PKC-mediated+hyperphosphorylation.">Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation.</a> Mol Cell. 28 (1), 68-78 (2007).</li><li>Kalsotra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mef2+transcription+network+is+disrupted+in+myotonic+dystrophy+heart+tissue,+dramatically+altering+miRNA+and+mRNA+expression.">The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression.</a> Cell Rep. 6 (2), 336-345 (2014).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270 (5639), 725-727 (1977).</li><li>Blau, H. M., Chiu, C. P., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+activation+of+human+nuclear+genes+in+stable+heterocaryons.">Cytoplasmic activation of human nuclear genes in stable heterocaryons.</a> Cell. 32 (4), 1171-1180 (1983).</li><li>Peng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celf1+regulates+cell+cycle+and+is+partially+responsible+for+defective+myoblast+differentiation+in+myotonic+dystrophy+RNA+toxicity.">Celf1 regulates cell cycle and is partially responsible for defective myoblast differentiation in myotonic dystrophy RNA toxicity.</a> Biochim Biophys Acta. 1852 (7), 1490-1497 (2015).</li><li>Amack, J. D., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myotonic+dystrophy+expanded+CUG+repeat+tract+is+necessary+but+not+sufficient+to+disrupt+C2C12+myoblast+differentiation.">The myotonic dystrophy expanded CUG repeat tract is necessary but not sufficient to disrupt C2C12 myoblast differentiation.</a> Hum Mol Genet. 10 (18), 1879-1887 (2001).</li><li>Amack, J. D., Paguio, A. P., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis+and+trans+effects+of+the+myotonic+dystrophy+(DM)+mutation+in+a+cell+culture+model.">Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model.</a> Hum Mol Genet. 8 (11), 1975-1984 (1999).</li><li>Bhagavati, S., Shafiq, S. A., Xu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(CTG)n+repeats+markedly+inhibit+differentiation+of+the+C2C12+myoblast+cell+line:+implications+for+congenital+myotonic+dystrophy.">(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: implications for congenital myotonic dystrophy.</a> Biochim Biophys Acta. 1453 (2), 221-229 (1999).</li><li>Amack, J. D., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+defects+in+myotonic+dystrophy.">Myogenic defects in myotonic dystrophy.</a> Dev Biol. 265 (2), 294-301 (2004).</li><li>Emerson, C. P., Sweeney, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in muscle biology. 52, (1997).</li><li>Ward, A. J., Rimer, M., Killian, J. M., Dowling, J. J., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CUGBP1+overexpression+in+mouse+skeletal+muscle+reproduces+features+of+myotonic+dystrophy+type+1.">CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1.</a> Hum Mol Genet. 19 (18), 3614-3622 (2010).</li><li>Koshelev, M., Sarma, S., Price, R. E., Wehrens, X. H. T., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart-specific+overexpression+of+CUGBP1+reproduces+functional+and+molecular+abnormalities+of+myotonic+dystrophy+type+1.">Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1.</a> Hum Mol Genet. 19 (6), 1066-1075 (2010).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="804">
	<tbody>
		<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 ...

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

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

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 ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides 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 ...