Name: Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells (Video) | JoVE Journal
Uploaded: 2016-07-29T14:00:00.000+00:00
Duration: 9 min 39 s
Description: Learn to establish myotonic dystrophy 1 models using C2C12 myoblast transfection and transduction techniques. This method enables investigation of muscular disease mechanisms and pathogenesis.

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5 minutes

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 cells. Place the plate in a 37 &#176;C, 5% CO2 incubator for 3 - 5 min.</li>
	<li>Neutralize the trypsin by adding 3 ml growth medium. Pipette 6 - 8 times to suspend the cells. Add 1 ml of suspended cell to a new 100 mm plate and incubate at 37 &#176;C, 5% CO2.</li>
</ol>
2. C2C12 Cell Transfection/transduction and Selection
<ol>
	<li>C2C12 plasmid transfection
	<ol>
		<li>24 hr prior to transfection, plate 7.0 x 105 C2C12 cells into one well of a six-well plate for each transfection. 
		Note: This leads to 50 - 60% confluence by the time of transfection.</li>
		<li>Allow the transfection reagent to come to room temperature prior to use.</li>
		<li>Add 2 &#181;g of plasmid DNA (GFP-CUG5 or GFP-CUG200) into 200 &#181;l pre-warmed serum free medium and vortex briefly. Add 6 &#181;l transfection reagent to the medium and mix immediately for 30 sec using a vortex. Incubate the mixture for 30 min at room temperature.</li>
		<li>Change the media of C2C12 cells to 2.5 ml serum-free growth media. Add the transfection mixture to the cells after incubation. Return the plates to a 5% CO2, 37 &#176;C incubator.</li>
		<li>Keep the cells in transfection mixture/serum-free growth media for 4 hr or up to overnight. Change media back to the growth media the next day or when cytotoxicity is apparent. 
		Note: Cytotoxicity is assessed by microscopic inspection of the cells. Observe the changes in cell morphology and cell detachment. As long as there is no overt cytotoxicity, overnight incubation in serum-free growth media enhances transfection.</li>
		<li>Select the cells 48 hr after transfection by adding 1.2 - 1.6 mg/ml G418 for ~ 10 days until the control culture (transfected without the plasmids) does not have live cells. Change the media every two days with growth media supplemented with G418.</li>
		<li>Maintain the G418 resistant cells in growth medium and 5% CO2 at 37 &#176;C for subsequent experiments.</li>
	</ol>
	</li>
	<li>Retrovirus preparation and C2C12 retroviral transduction
	<ol>
		<li>Prepare HEK 293 culture medium by supplementing DMEM high glucose with 10% fetal bovine serum, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, and 2 mM L-glutamine.
		<ol>
			<li>Approximately 24 hr prior to transfection, plate 4.0 x 106 ecotropic HEK 293-based packaging cells in a 100 mm plate (80% confluence at transfection) containing cell culture medium.</li>
		</ol>
		</li>
		<li>Allow the transfection reagent to come to room temperature prior to use.</li>
		<li>Mix 15 &#181;g plasmid DNA (pMSCV-Puro or pMSCV-Celf1Flag-Puro) in 1 ml serum-free growth medium. Vortex briefly. Add 30 &#181;l transfection reagent from step 2.2.2 into the DNA/serum-free growth medium tube, and mix well using vortex. Incubate the mixture at room temperature for 30 min.</li>
		<li>Add transfection mixture dropwise to the ecotropic HEK 293-based packaging cells. Gently swirl the plates and return them back to the 5% CO2, 37 &#176;C incubator. After 24 hr, change the media to 10 ml pre-warmed fresh growth medium.</li>
		<li>Harvest the supernatant (containing retrovirus) 48 hr after transfection using a pipette and transfer the supernatant into a 50 ml centrifuge tube. Add 10 ml of new warm media to the plate and return it to the incubator. Keep the supernatant at 4 &#176;C. 
		Note: The media is added gently to the side of the well so as not to disrupt the cell monolayer.</li>
		<li>Harvest the second batch of supernatant 60 hr after transfection and pool it with the supernatant from 2.2.5. Centrifuge at 500 x g, 4 &#176;C for 7 min to remove cellular debris.</li>
		<li>Transfer the supernatant into a new 50 ml centrifuge tube. Use retrovirus to transduce C2C12 cells at this point by proceeding to step 2.2.9 or aliquot and store the supernatant at -20 &#176;C for future use.</li>
		<li>Thaw the retrovirus from 2.2.7 at room temperature, if a frozen stock is used here. 
		Note: Avoid multiple freeze-thaw cycles.</li>
		<li>Plate C2C12 cells on the same day of transduction aiming for 30 - 40% of confluence.
		<ol>
			<li>Discard the growth medium and wash C2C12 cells with 3 ml room temperature PBS. Remove the PBS, add 500 &#181;l 0.25% Trypsin-EDTA, and incubate the plate in a 37 &#176;C, 5% CO2 incubator for 3 - 5 min.</li>
			<li>Neutralize the trypsin by adding 3 ml growth medium. Pipette 6 - 8 times to suspend the cells. Count cells using a hemocytometer and add 8.0 x 105 C2C12 cells to a 60 mm plate.</li>
		</ol>
		</li>
		<li>Add 0.5 ml retrovirus to infect one 60 mm plate of cells in 3 ml growth media. Return the plate to the incubator.</li>
		<li>Replace with 3 ml fresh media supplemented with selection antibiotic (puromycin 1 - 3 &#181;g/ml) 48 hr after infection. Replace 3 ml medium with antibiotic every day for ~ 5 days until the untransduced C2C12 control culture dies out.</li>
	</ol>
	</li>
	<li>Lentivirus preparation and C2C12 lentiviral transduction 
	Note: Perform all the lentivirus-related procedures in Biosafety Level 2 cabinet and follow biological safety guidelines. Liquid waste containing the virus must be disinfected prior to disposal.
	<ol>
		<li>Plate 4.0 x 106 293T cells in a 100 mm plate (~ 80% confluence at day of transfection) with HEK 293 cell culture medium (DMEM high glucose, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, and 2 mM L-glutamine) 24 hr before transfection.</li>
		<li>Warm the transfection reagent to room temperature. Mix the lentiviral packaging vectors (4 &#181;g pMD2.G and 6 &#181;g psPAX2) together with 8 &#181;g lentiviral transfer vector (Celf1 shRNA or scrambled control shRNA) in 1 ml serum free cell culture medium. Vortex briefly.</li>
		<li>Add 36 &#181;l pre-warmed transfection reagent to the DNA/DMEM tube and mix well by vortex. Incubate the mixture at room temperature for 30 min.</li>
		<li>Remove the 293T cells from the incubator and add the transfection mixture to the plate. Gently swirl the plate and return it back to 5% CO2, 37 &#176;C incubator. After 24 hr, replace the transfection mixture with 10 ml fresh culture medium.</li>
		<li>Collect the supernatant containing lentivirus 48 hr after transfection and transfer it into a 50 ml centrifuge tube. Add 10 ml new warm media to the plate and return it to incubator. Store the supernatant at 4 &#176;C.</li>
		<li>Collect the second batch of supernatant 60 hr after transfection and combine it with the supernatant from 2.3.5. Centrifuge briefly at 500 x g, 4 &#176;C for 7 min.</li>
		<li>Transfer the supernatant into a fresh 50 ml centrifuge tube. If using fresh virus, proceed to step 2.3.9. For future use, store virus-containing supernatant at -20 &#176;C in aliquots of 10 ml.</li>
		<li>Thaw the virus from 2.3.7 at room temperature before use, if a frozen stock is used. 
		Note: Avoid multiple freeze-thaw cycles.</li>
		<li>Plate C2C12-CUG200 cells (obtained in Section 2.1) as described in 2.2.9 on the same day of transduction. 
		Note: Aim for 30 - 40% of confluence.</li>
		<li>Add 0.5 ml virus to infect a 60 mm plate of C2C12-CUG200 and return the plate to the incubator.</li>
		<li>Replace the culture medium with fresh media supplemented with the selection antibiotic (puromycin 1 - 3 &#181;g/ml) 72 hr after transduction. Refresh culture media everyday with the selection antibiotic for ~5 days until all untransduced C2C12-CUG200 control culture dies out.</li>
		<li>Use Western blot to verify Celf1 knockdown in GFP-CUG200 transfected cells.</li>
	</ol>
	</li>
</ol>
3. C2C12 Cell Differentiation
<ol>
	<li>Plate 2 x 106 cells in one well of a 6-well plate in 2.5 ml growth media 24 hr before initiation of differentiation. 
	Note: The plating density may be adjusted so that full confluence is reached in 24 hr.
	<ol>
		<li>Rinse confluent cells with PBS and add 2.5 ml of low-serum differentiation medium (Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 2% equine serum, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 2 mM L-glutamine, and 1 &#181;M insulin). Replace the medium every two days. 
		Note: Keep stock insulin frozen and add it to the media at each medium change.</li>
	</ol>
	</li>
	<li>During C2C12 differentiation, collect samples for quantitative RT-PCR (see section 4) at the following time points: Day0, 1, 2, 4, and 6. Process the culture for immunostaining at day 6 - 8 (see section 5).</li>
</ol>
4. RNA Isolation and Quantitative RT-PCR
<ol>
	<li>Use the manufacturer&#39;s protocol for RNA isolation. After isolation, resuspend the RNA pellet in 30 &#181;l RNase-free water and pipet up and down several times. 
	Note: The samples may proceed to Quantitative RT-PCR or can be stored at -80 &#176;C.</li>
	<li>Use a RT qPCR kit for Quantitative RT-PCR10 and follow the manufacturer&#39;s instruction. Prepare the reaction mix according to Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;l)</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td>2x reaction buffer</td>
			<td>10</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Forward primer</td>
			<td>0.5</td>
			<td>200 nM</td>
		</tr>
		<tr>
			<td>Reverse primer</td>
			<td>0.5</td>
			<td>200 nM</td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>0.5</td>
			<td>200 nM</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase &#38; RNase Inhibitor</td>
			<td>0.1</td>
			<td>0.25 U/ml</td>
		</tr>
		<tr>
			<td>Template</td>
			<td>1</td>
			<td>100 ng total RNA</td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td>7.4</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Total Mix</td>
			<td>20</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Quantitative RT-PCR Master Mix Preparation
<ol start="3">
	<li>Run a 20 &#181;l RT-qPCR reaction using thermal profile Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reverse transcription step</td>
			<td>30 min, 48 &#176;C</td>
		</tr>
		<tr>
			<td>DNA polymerase activation/Reverse Transcriptase inactivation</td>
			<td>10 min, 95 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">40 Cycles</td>
			<td>15 sec, 95 &#176;C</td>
		</tr>
		<tr>
			<td>1 min, 60 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 2. Quantitative RT-PCR thermal profile
5. Immunostaining
<ol>
	<li>Fix the monolayer culture in 2.5 ml freshly prepared 4% paraformaldehyde/PBS at room temperature for 10 min.</li>
	<li>Permeabilize the samples in 2.5 ml 0.1% Triton X-100/PBS for 30 min at room temperature.</li>
	<li>Transfer the samples to a humidified chamber. Block the samples in 2.5 ml blocking buffer (0.1% nonionic surfactant/PBS, 10% normal goat serum) for 30 min at 37 &#176;C.</li>
	<li>Discard blocking buffer and apply 800 &#181;l primary antibody against Myosin Heavy Chain diluted in blocking buffer (1:100). Incubate in a humidified chamber overnight at room temperature.</li>
	<li>Wash the plate 3 times (10 min each) with 2.5 ml wash buffer (0.1% polysorbate 20/PBS) on day 2.</li>
	<li>Remove the wash buffer and apply 800 &#181;l secondary antibody (goat anti-mouse IgG, 1:500 diluted in PBS). Incubate in a humidified chamber at room temperature for 1 hr.</li>
	<li>Wash two times (10 min each) with 2.5 ml wash buffer.</li>
	<li>Incubate with 800 &#181;l DAPI (1 &#181;g/ml, diluted in distilled deionized H2O) for 5 min.</li>
	<li>Wash with 2.5 ml wash buffer again for 10 min.</li>
	<li>Change the washing buffer to 2.5 ml PBS and use a fluorescent microscope to capture images.</li>
</ol>

<ol><li>Harper, P. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHarper%2C+PS+%22Myotonic+dystrophy.+3rd+edn%22">Myotonic dystrophy. 3rd edn</a>. , W. B. Saunders. (2001).</li>
<li>Timchenko, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+of+muscle+atrophy+in+myotonic+dystrophies%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Molecular+basis+of+myotonic+dystrophy%3A+expansion+of+a+trinucleotide+%28CTG%29+repeat+at+the+3%27+end+of+a+transcript+encoding+a+protein+kinase+family+member%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Developmental+insights+into+the+pathology+of+and+therapeutic+strategies+for+DM1%3A+Back+to+the+basics%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">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/pubmed?term=((Muscleblind+proteins+regulate+alternative+splicing%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Increased+steady-state+levels+of+CUGBP1+in+myotonic+dystrophy+1+are+due+to+PKC-mediated+hyperphosphorylation%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">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/pubmed?term=((The+Mef2+transcription+network+is+disrupted+in+myotonic+dystrophy+heart+tissue%2C+dramatically+altering+miRNA+and+mRNA+expression%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">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/pubmed?term=((Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Cytoplasmic+activation+of+human+nuclear+genes+in+stable+heterocaryons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Celf1+regulates+cell+cycle+and+is+partially+responsible+for+defective+myoblast+differentiation+in+myotonic+dystrophy+RNA+toxicity%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">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/pubmed?term=((The+myotonic+dystrophy+expanded+CUG+repeat+tract+is+necessary+but+not+sufficient+to+disrupt+C2C12+myoblast+differentiation%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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/pubmed?term=((Cis+and+trans+effects+of+the+myotonic+dystrophy+%28DM%29+mutation+in+a+cell+culture+model%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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/pubmed?term=((%28CTG%29n+repeats+markedly+inhibit+differentiation+of+the+C2C12+myoblast+cell+line%3A+implications+for+congenital+myotonic+dystrophy%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">(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/pubmed?term=((Myogenic+defects+in+myotonic+dystrophy%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aEmerson%2C+CP+%22Methods+in+muscle+biology%22">Methods in muscle biology</a>. 52, Academic Press. (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/pubmed?term=((CUGBP1+overexpression+in+mouse+skeletal+muscle+reproduces+features+of+myotonic+dystrophy+type+1%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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/pubmed?term=((Heart-specific+overexpression+of+CUGBP1+reproduces+functional+and+molecular+abnormalities+of+myotonic+dystrophy+type+1%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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><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

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 ± 4.1% to 2.6 ± 1.1% and myotube areas were decreased from...

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 ...

The overall goal of these myoblast maintenance, transfection, and transduction procedures is to establish a myotonic dystrophy one cell model for the study of myotonic dystrophy and other muscular diseases. This method can help establish the M1 myoblast cell models that will be useful in investigating the detailed mechanism of the M1 pathogenesis. This multi tiered gene manipulation of C2C12 myoblasts enables the modeling of sequential and genetic pathological events in the M1.24 hours before their transfection plate seven times 10 to the fifth C2C12 cells per well in a six well plate.The next day add two micro grams of the plasmid DNA of interest into 200 micro liters of room temperature, serum free, medium and briefly vortex the solution. Next, add six micro liters of room temperature transfection reagent to the medium, and immediately vortex the solution for 30 seconds. Then incubate the medium at room temperature for 30 minutes.During the last five minutes of the incubation. Replace the medium in each well with two point five milliliters of fresh serum free growth medium, and add 200 micro liters of transfection mixture to each well. Return the plate to the cell culture incubator for four hours.Then check the cells under a microscope for changes in their morphology and attachment. If cytotoxicity is observed, replace the serum free medium with growth medium. 48 hours after transfection, add the appropriate selection antibiotic in fresh growth medium every two days for 10 days.Until the control transfected without plasmids well does not contain live cells. To prepare the retrovirus, first plate four times 10 to the sixth ecotropic HEK293 based packaging cells in a 100 millimeter plate containing cell culture medium. The next day, mix 15 micro grams of the plasmid DNA of interest in one milliliter of serum free growth medium, and briefly vortex the tube.Next, add 30 micro liters of room temperature transfection reagent to the medium, and immediately vortex the solution for 30 seconds. After 30 minutes at room temperature, add the transfection mixture drop wise to the ecotropic cells, and gently swirl the plate. Return the plate to the cell culture incubator for 24 hours, replacing the medium with 10 milliliters of 37 degree celsius fresh growth medium the next day.48 hours after the transfection, use a pipet to transfer the retro viral containing supernatants into a single 50 milliliter conical tube for overnight storage at four degrees Celsius. Then add 10 milliliters of new warm medium down the side of the plate, and return the culture to the incubator. Harvest the second batch of retro virus 60 hours after the transfection.Pooling it with the retrovirus collected at 48 hours. Spin down the supernatant to remove any cellular debris, and transfer the retrovirus into a new 50 milliliter tube. Now wash each well of a C2C12 cell culture with three milliliters of room temperature PPS followed by the addition of 500 micro liters of zero point two five percent trypsin EDTA for a five minute incubation at 37 degrees Celsius.When the cells have detached, neutralize the reaction in each well with three milliliters of growth medium and triturate the culture six to eight times to disassociate the cells into a single cell suspension. After counting add eight times 10 to the fifth cells to a 60 milliliter plate, and add three milliliters of growth medium. Then infect the cells with zero point five milliliters of retro virus and return the plate to the incubator for 48 hours.After two days, feed the cells with three milliliters of fresh growth medium, supplemented with the appropriate selection antibiotic everyday for five days. Until the untransduced C2C12 control culture contains no more living cells. For C2C12 cell lentiviral transduction, in a bio-safety level two cabinet, plate four times 10 to the sixth 293T cells in a 100 millimeter plate with HEK293 cell culture medium.The next day, mix the lenitviral packaging vectors with eight micro grams of the appropriate lenitviral transfer vector, in one milliliter of serum free cell culture medium. Vortex the mixture briefly and add 36 micro liters of pre-warmed transfection reagent. Then vortex the medium for another 30 seconds, and incubate the mixture at room temperature for 30 minutes.At the end of the incubation, add the transfection mixture to the 293T cells, gently swirl the plate, and return the cells to the incubator. After 24 hours, replace the transfection mixture with 10 milliliters of fresh culture medium. After 48 hours, transfer the lentivirus containing supernatant into a 50 milliliter conical tube for four degree celsius storage, and add 10 milliliters of fresh, warm medium to the plate.After 60 hours, pool the second batch of supernatant with the lentivirus collected at 48 hours, and centrifuge the virus to remove any cell debris. Then add zero point five milliliters of lentivirus to a 30 to 40 percent confluent culture of C2C12 CUG200 cells seated in a 60 millimeter plate as just demonstrated. 72 hours after transfection, replace the lentivirus supernatant with fresh medium, supplemented with the appropriate selection antibiotic.Refreshing the culture medium every day for five days with fresh selection antibiotic supplemented medium, until all of the untransduced C2C12 CUG200 control cells are dead. After drug resistance selection, the GFP expression and myotube formation of differentiated myobalsts can be assessed by a manofluorescent analysis. Quantification of the myotube formation in this representative experiment demonstrates that the fusion indecies and myotube areas are decreases significantly by abnormal CUG expansion and GFP CUG 200 transfected cells.Celf1 upregulation is also associated with CUG expansion in myogenic dystrophy one. Further, western blotting consistently demonstrates and elevation of Celf1 protein levels with CUG inhibited expression of MyoD, MyoG, and Mef2C during differentiation. Suggesting that CUG expansion induces myotube defects and impaired myoblast differentiation.Western blot analysis also reveals both that FLAG-tagged Celf1 is present only in retroviral vector transduced cultures, and that it's expression is up regulated in these cultures. Indeed myotube formation is rare in Celf1 over-expressing cells compared to control cultures. Suggesting that the over-expression of Celf1 severely impairs myoblast differentiation.Further, the level of endogenous Celf1 protein and double antibiotic selected lentiviral vector transduced GFP CUG 200 cells is markedly reduced in the presence of Celf1 short hairpin RNA. With the rescue of myotube formation, myotube fusion indecies and myotube areas observed in these cultures. Meanwhile, real time RTPCR results suggest that the expression of MyoD, MyoG, and Mef2C are significantly increased in Celf1 short hairpin RNA expressing cells supporting the possibility of a Celf1 knock down rescued myocyte differentiation deficiency in these cultures.While attempting this procedure, it's important to monitor the C2C12 cell density to be below 70 percent confluence. Following the cell transfection, treat the cells with serum free medium for at least four hours, but no more than 12 hours. While monitoring the cells cytotoxicity.Through the use of plasmid transfection, retroviral and lentiviral transduction, we have successfully established a myotonic dystrophy cell model which will benefit research in this and other muscular diseases.

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

Research

JoVE Journal

Medicine

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

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Learn to establish myotonic dystrophy 1 models using C2C12 myoblast transfection and transduction techniques. This method enables investigation of muscular disease mechanisms and pathogenesis.

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

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