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>

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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. 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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 transfection/transduction and maintenance of consistency in differentiation are highlighted below.
The cell density in daily cell maintenance is critical. These cells need to be cultured below 50 - 70% confluence. Due to the short population doubling time, high confluence (&#62; 70%) C2C12 culture spontaneously differentiates when left overnight. While the fraction of spontaneously differentiated cells may be small, the presence of differentiated cells will contribute to inconsistencies in subsequent differentiation experiments. Additionally, freshly thawed C2C12 cells that have low passage number are preferred. For differentiation, cells are plated at pre-determined numbers so that they reach full confluence after 24 hr. Insulin is kept frozen in small aliquots and is added each time the differentiation medium is changed. These measures improve differentiation outcomes.
Serum free medium enhances C2C12 cell transfection. The cells can be incubated in transfection mixture/serum free media overnight. In our lab, we achieve up to 20% transfection rate, accompanied by slight signs of cytotoxicity. The concentration of drug used to select C2C12 cell is higher, 1.2 to 1.6 mg/ml G418 or 1 to 3 &#181;g/ml puromycin, and the duration of the selection is longer than most other cell lines. Due to potential toxicity under high drug concentration and the extended incubation period, lot-to-lot variation in differentiation potential may arise. It is crucial to adjust the selection scheme (concentration and duration) based on cell appearance and to treat the experiment and control groups together.
We have successfully introduced GFP-CUG200 into C2C12 cells that reproduce 3&#39;-UTR CUG expansion in DM1. Introducing Celf1 into C2C12 cells and Celf1 shRNA into GFP-CUG200 C2C12 cells has allowed the evaluation of cellular and molecular events triggered by Celf1 upregulation and exploration of targeting Celf1 to help DM1 myoblast differentiation, respectively. In summary, the C2C12 myoblast model presented here benefits the investigation of myotonic dystrophy as well as general muscle cell biology and myogenic differentiation. The limitation of this model is that the findings in cell lines may not completely extend to organisms. The findings in the current models are often advanced to isolating fresh myoblasts from animals and studying their differentiation. Ultimately we substantiate myoblast findings in organisms.
The unique strength of this study is the multi-tier gene manipulation in C2C12 myoblasts, which enables modeling the sequential genetic/pathological events in DM1. Previously, the RNA toxicity effect of DMPK CUG expansion was documented in myoblasts11-14, while the pathological roles of Celf1 were separately studied in mouse models16,17. Modeling these sequential genetic/pathological events is technically challenging and time consuming if performed in animal models. As demonstrated in this study, the combined use of fluorescent and drug resistant markers allows the modeling of additional molecular events in myotonic dystrophy. The models established in this study would be useful in dissecting the detailed mechanisms in DM1 pathogenesis. They would also be valuable in screening for drugs that target different steps of DM1 pathogenesis. The strategies and procedures of this study may shed light on establishing other muscular disease models in myoblasts.

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

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 decreased from 35.6 &#177;&#160; 2.2% to 2.7 &#177; 0.8% by abnormal CUG expansion in GFP-CUG200 (Figure 1C). The fusion index and the myotube area were counted via the total number of nuclei in myotubes (&#8805; 2 nuclei) divided by the total number of nuclei and the percentage of the total image area covered by myotubes, respectively. Real-time RT-PCR10 was conducted to analyze expression for a number of muscle differentiation related genes including MyoD, MyoG, Mef2c and Celf1. Compared to CUG5 cultures, CUG200 increased the expression of Celf1 mRNA in proliferating myoblasts at the beginning of differentiation. Congruent with previous reports, Celf1 upregulation was associated with CUG-expansion in myogenic dystrophy 1 (Figure 1D). Further, Western blotting10 consistently showed Celf1 protein level was elevated (Figure 1E), and CUG200 inhibited the expression of MyoD, MyoG and Mef2c during differentiation (Figure 1D). These results suggest that the CUG-expansion leads to myotube defects and impaired myoblast differentiation.
To study the role of Celf1 in myoblast differentiation, pMSCV- Celf1Flag retroviral vector was constructed to transduce C2C12 cells.10 Puromycin-resistant clones were pooled for differentiation studies. Western blot results showed that the Flag-tagged Celf1 was only present in the pMSCV-Celf1Flag transduced cultures and the expression of Celf1 protein was upregulated (Figure 2A). The formation of myotubes in Celf1-overexpressing cells is rare when compared to the control cultures (Figure 2B), which suggests that the overexpression of Celf1 severely impairs myoblast differentiation.
To determine if Celf1 knockdown rescues the differentiation deficiency in CUG-expansion cells, Celf1 shRNA was delivered to GFP-CUG200 cells by lentiviral vectors. Double resistant clones (G418 and puromycin) were selected and pooled for differentiation studies. The level of endogenous Celf1 protein was markedly reduced in the presence of Celf1 shRNA (Figure 3A). After 6 days of differentiation, the effect of Celf1 shRNA on increasing myotube formation was evident (Figure 3B). Fusion indices and myotube areas were 16.1 &#177; 3.0% and 15.2 &#177; 1.2%, respectively, compared to 4.6 &#177; 0.8% and 5.1 &#177; 1.3% in control cultures (Figure 3C). Meanwhile, real-time RT-PCR results suggest that the expression of MyoD, MyoG and Mef2c was significantly increased in Celf1 shRNA cells (Figure 3D) supporting Celf1 knockdown rescued myocyte differentiation deficiency.
<img alt="Figure 1" src="/files/ftp_upload/54078/54078fig1.jpg" /> 
Figure 1. CUG-Expansion Inhibits Myocyte Differentiation in C2C12 Cells. (A) C2C12 cells expressed GFP-CUG5 or GFP-CUG200 ubiquitously after selection. Scale bars are 100 &#181;m. This figure has been modified from our previous paper10. (B) Myotubes were formed in GFP-CUG5 cells but less in GFP-CUG200 cells. Myotubes were visualized by myosin heavy chain (MF-20) immunostaining. Scale bars are 100 &#181;m. (C) GFP-CUG200 cultures significantly reduced fusion index and myotube areas. The error bars represent standard deviations. (D) Real-time RT-PCR analysis of Celf1, transcription factors MyoD, MyoG, and Mef2c expression during myoblast differentiation. mRNA levels were plotted after the GAPDH normalized. The error bars represent standard deviations. Student&#39;s T test was performed to compare the tests to controls. n &#62; 3, *P &#60; 0.05. (E) GFP-CUG200 transfection increased Celf1 protein expression. &#223;-actin is shown as the loading control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54078/54078fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54078/54078fig2.jpg" /> 
Figure 2. Overexpression of Celf1 Impairs Myoblast Differentiation. (A) Proof of exogenous Celf1Flag expression in C2C12 cells by Western blot. (B) Myotube formation was impaired in Celf1Flag-overexpressing C2C12 myoblasts. Myotubes were visualized by MF-20 immunostaining. Scale bars are 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54078/54078fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54078/54078fig3.jpg" /> 
Figure 3. Knockdown of Celf1 Partially Rescues CUG-Expansion-Induced Myoblast Differentiation Deficiency. (A) Proof of Celf1 knockdown by Western blot. (B) Celf1 shRNA induced myotube formation in GFP-CUG200 myoblasts. Myotubes were visualized by MF-20 immunostaining. Scale bars are 100 &#181;m. (C) Celf1 shRNA-rescued cells that had increased fusion index and myotube areas. The error bars represent standard deviations. (D) Real-time RT-PCR analysis of Celf1, transcription factors MyoD, MyoG, and Mef2c expression during differentiation. mRNA level was plotted after normalizing to that of GAPDH. The error bars represent standard deviations. Student&#39;s T test was performed to compare the tests to control. n &#62; 3, *P &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54078/54078fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

15.3K Views.  University of Houston.  In dit protocol presenteren we de procedures vaststelling myotone dystrofie 1 myoblast modellen, waaronder geoptimaliseerde C2C12 celonderhoud, gen transfectie / transductie en myocyt differentiatie. 

Video: Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Bioengineering

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Developmental Biology

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

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

Neuroscience

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

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

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

Genetics

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

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

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

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

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

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