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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	<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. 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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. 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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 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 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/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/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/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

15.1K Views.  University of Houston. 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.

Video: Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

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

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

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

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

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

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

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

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

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

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

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

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

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

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

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

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

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

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

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

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

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

Modeling Chemotherapy Resistant Leukemia In Vitro

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

Modeling Chemotherapy Resistant Leukemia In Vitro

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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