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 Cell Culture
<ol>
	<li>Maintain C2C12 mouse myoblasts in a 100 mm plate in growth medium (Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)) supplemented with 20% fetal bovine serum, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, and 2 mM L-glutamine. Allow C2C12 passaged cells to become approximately 50 - 60% confluent.</li>
	<li>Discard the growth medium and wash C2C12 cells with 3 ml room temperature phosphate-buffered saline (PBS). Remove the PBS and add 500 &#181;l 0.25% Trypsin-EDTA to detach the cell...

<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>

C2C12 cell line has been used as a model to study myogenesis11-14. These cells retain a fibroblast-like look, proliferate rapidly in media containing 20% fetal bovine serum and readily differentiate in media containing 2% equine serum15. The fast growth and differentiation are advantageous characteristics in a myogenesis cell model. Here, we demonstrate the use of plasmid, retroviral, and lentiviral vectors to introduce cDNA, 3&#39;-UTR, and shRNA into C2C12 cells. The critical points for transfecti...

Myotonic dystrophy (DM) is an autosomal dominant disease that affects multiple systems, most notably cardiac and skeletal muscles1. There are two subtypes of this disease, DM1 and DM2. DM1 is more common and has a more severe manifestation than DM22. The genetic mutation underlying DM1 is an expansion of CUG triplet repeats located in the 3&#39; untranslated region (UTR) of DM protein kinase gene (DMPK)3. The CUG repeat number in unaffected individuals varies from 5 to 37. In contrast, it increases to more than 50, and sometimes up to thousands in DM1 patients4. As a result, RNA-binding proteins, such as muscleblind-like 1 (MBNL1), CUGBP, and Elav-like family 1 (Celf1), are misregulated. Due to the sequestration on the expanded CUG repeats, MBNL1 loses its ability to regulate alternative splicing5. Celf1, on the other hand, is up-regulated6,7. Overexpression of Celf1 is associated with muscle loss and weakness, which are not attributed to loss of MBNL1 function. Animal models simulating DM1-related alterations, including DMPK 3&#39;-UTR CUG expansion, loss of MBNL1, and overexpression of Celf1, have been established. However, modeling DM1 in myoblasts offers an efficient alternative, especially for dissecting DM1-related cellular and molecular events.
C2C12 myoblast cell line was first isolated from injured C3H mouse muscle and widely used to study myogenic differentiation8,9. C2C12 cells rapidly proliferate in fetal bovine serum (FBS)-containing media and readily undergo differentiation when FBS is depleted. Yet, using this myoblast differentiation model presents two challenges: C2C12 cells are often resistant to gene transfection/viral transduction; and slight variations in cell handling and differentiation procedure can lead to marked changes in myotube formation.
Our lab routinely uses C2C12 myoblasts as a cell model and has developed protocols that effectively deliver genes by plasmid transfection, retroviral transduction, and lentiviral transduction into C2C12 cell line10. In the video, we demonstrate the optimized procedures for transfecting/transducing C2C12 cells and maintaining differentiation consistency in establishing DM1 myoblast models.

<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

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

C2C12 cells were transfected with GFP-CUG5 or GFP-CUG200. After drug-resistance selection, stable pools were established, which can be visualized by GFP expression (Figure 1A). Myotube formation in the differentiated myoblasts was detected by myosin heavy chain immunostaining10 (Figure 1B). The quantification of myotube formation demonstrated that fusion indices were decreased from 35.4 &#177; 4.1% to 2.6 &#177; 1.1% and myotube areas were decr...

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 this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

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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