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

We thank Drs. Tom Cooper from the Baylor College of Medicine, Mani S. Mahadevan from the University of Wisconsin-Madison, and Didier Trono from the University of Geneva for reagents. This work is supported by a University of Houston startup fund (YL), American Heart Association grant (YL, 11SDG5260033), and the National Natural Science Foundation of China (XP, 81460047).

1. C2C12 Cell Culture <ol><li> Onderhouden C2C12 muis myoblasten in 100 mm platen in kweekmedium (Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM)) aangevuld met 20% foetaal bovien serum, 100 U / ml penicilline, 100 ug / ml streptomycine en 2 mM L-glutamine. Sta C2C12 cellen gepasseerd tot ongeveer 50 worden - 60% confluent. </li><li> Gooi het groeimedium en was C2C12 cellen met 3 ml kamertemperatuur fosfaatgebufferde zoutoplossing (PBS). Verwijder de PBS en voeg 500 ul 0,25% trypsine-EDTA op de cellen los. Plaa...

<ol>
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M., Chiu, C. P., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+activation+of+human+nuclear+genes+in+stable+heterocaryons.">Cytoplasmic activation of human nuclear genes in stable heterocaryons.</a> Cell. 32 (4), 1171-1180 (1983).</li><li>Peng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celf1+regulates+cell+cycle+and+is+partially+responsible+for+defective+myoblast+differentiation+in+myotonic+dystrophy+RNA+toxicity.">Celf1 regulates cell cycle and is partially responsible for defective myoblast differentiation in myotonic dystrophy RNA toxicity.</a> Biochim Biophys Acta. 1852 (7), 1490-1497 (2015).</li><li>Amack, J. D., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myotonic+dystrophy+expanded+CUG+repeat+tract+is+necessary+but+not+sufficient+to+disrupt+C2C12+myoblast+differentiation.">The myotonic dystrophy expanded CUG repeat tract is necessary but not sufficient to disrupt C2C12 myoblast differentiation.</a> Hum Mol Genet. 10 (18), 1879-1887 (2001).</li><li>Amack, J. D., Paguio, A. P., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis+and+trans+effects+of+the+myotonic+dystrophy+(DM)+mutation+in+a+cell+culture+model.">Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model.</a> Hum Mol Genet. 8 (11), 1975-1984 (1999).</li><li>Bhagavati, S., Shafiq, S. A., Xu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(CTG)n+repeats+markedly+inhibit+differentiation+of+the+C2C12+myoblast+cell+line:+implications+for+congenital+myotonic+dystrophy.">(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: implications for congenital myotonic dystrophy.</a> Biochim Biophys Acta. 1453 (2), 221-229 (1999).</li><li>Amack, J. D., Mahadevan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+defects+in+myotonic+dystrophy.">Myogenic defects in myotonic dystrophy.</a> Dev Biol. 265 (2), 294-301 (2004).</li><li>Emerson, C. P., Sweeney, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in muscle biology. 52, (1997).</li><li>Ward, A. J., Rimer, M., Killian, J. M., Dowling, J. J., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CUGBP1+overexpression+in+mouse+skeletal+muscle+reproduces+features+of+myotonic+dystrophy+type+1.">CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1.</a> Hum Mol Genet. 19 (18), 3614-3622 (2010).</li><li>Koshelev, M., Sarma, S., Price, R. E., Wehrens, X. H. T., Cooper, T. 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C2C12 cellijn is gebruikt als een model om myogenesis 11-14 bestuderen. Deze cellen behouden een fibroblast-achtige look, prolifereren snel in media die 20% foetaal runderserum en gemakkelijk differentiëren in media die 2% paarden serum 15. De snelle groei en differentiatie zijn voordelige eigenschappen in een myogenesis cel model. Hier tonen we het gebruik van plasmide, retrovirale en lentivirale vectoren om cDNA, 3&#39;-UTR en shRNA introduceren in C2C12-cellen. De kritische punten voor transfec...

Myotone dystrofie (DM) is een autosomaal dominante ziekte die meerdere systemen, met name hart- en skeletspieren 1 beïnvloedt. Er zijn twee subtypen van deze ziekte, DM1 en DM2. DM1 is gebruikelijker en een ernstiger uiting dan DM2 2. De genetische mutatie onderliggende DM1 is een uitbreiding van CUG triplet repeats in het 3 &#39;onvertaalde gebied (UTR) van DM proteïne kinase gen (DMPK) 3. De CUG aantal repeats in aangetaste individuen varieert van 5 tot 37. Daarentegen verhoogt tot meer dan 50 en soms wel duizenden in DM1 patiënten 4. Hierdoor RNA-bindende eiwitten, zoals muscleblind-like 1 (MBNL1), CUGBP en Elav-achtige familie 1 (Celf1), zijn misregulated. Door de opslag van de geëxpandeerde CUG herhalingen, verliest MBNL1 zijn vermogen om alternatieve splicing 5 reguleren. Celf1, anderzijds, wordt opwaarts gereguleerd 6,7. Overexpressie van Celf1 gaat gepaard met het verlies van spiermassaen zwakheid, die niet worden toegeschreven aan verlies van MBNL1 functie. Diermodellen simuleren DM1-gerelateerde veranderingen, waaronder DMPK 3&#39;-UTR CUG expansie, verlies van MBNL1 en overexpressie van Celf1, zijn vastgesteld. Echter, modelleren DM1 in myoblasten biedt een efficiënt alternatief, vooral voor ontleden DM1-gerelateerde cellulaire en moleculaire gebeurtenissen. C2C12 myoblasten-cellijn werd voor het eerst geïsoleerd uit gewond C3H muis spier en op grote schaal gebruikt om myogene differentiatie 8,9 bestuderen. C2C12 cellen sneller prolifereren in foetaal bovine serum (FBS) bevattende media en gemakkelijk differentiatie ondergaan wanneer FBS leeg. Maar met deze myoblast differentiatie model presenteert twee problemen: C2C12 cellen zijn vaak resistent tegen gentransfectie / virale transductie; en lichte variaties in cel handling en differentiatie procedure kan leiden tot duidelijke veranderingen in myotube formatie. Ons laboratorium maakt gebruik van routinematig C2C12 myoblasten als acell model en heeft protocollen die effectief genen door plasmide transfectie, retrovirale transductie, en lentivirale transductie geven in C2C12 cellijn 10 ontwikkeld. In de video, tonen we de geoptimaliseerde procedures voor het transfecteren / transduceren C2C12 cellen en het behoud van differentiatie consistentie bij de vaststelling van DM1 myoblast modellen.

<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

Myotone dystrofie 1 (DM1) is een veel voorkomende vorm van spierdystrofie. Hoewel verschillende diermodellen vastgesteld voor DM1, myoblast cell modellen blijven belangrijk omdat ze een efficiënte cellulaire alternatief voor het bestuderen van cellulaire en moleculaire gebeurtenissen. Hoewel C2C12 myoblast cellen zijn op grote schaal gebruikt myogenesis, resistentie te bestuderen gen transfectie of virale transductie, belemmert onderzoek C2C12 cellen. We beschrijven hier een geoptimaliseerd protocol dat dagelijks onderhoud, transfectie en transductie procedures omvat om genen in C2C12-myoblasten en de inductie van differentiatie myocyten. Tezamen zijn deze procedures inschakelen beste transfectie / transductie efficiëntie en samenhangende differentiatie resultaten. Het protocol bij de vaststelling DM1 myoblasten celmodellen zou de studie van myotone dystrofie, evenals andere spierziekten profiteren beschreven.

C2C12-cellen werden getransfecteerd met GFP-CUG5 of GFP-CUG200. Na resistentie selectie werden stabiele pools opgericht, die worden gevisualiseerd kan door GFP expressie (Figuur 1A). Myotube formatie in het gedifferentieerde myoblasten werd gedetecteerd door immunokleuring myosine zware keten 10 (figuur 1B). De kwantificering van myotube formatie aangetoond dat fusie indices werden afgenomen van 35,4 ± 4,1% tot 2,6 ± 1,1% en myotube gebieden...

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 dit protocol presenteren we de procedures vaststelling myotone dystrofie 1 myoblast modellen, waaronder geoptimaliseerde C2C12 celonderhoud, gen transfectie / transductie en myocyt differentiatie.

Modelleren van myotone dystrofie 1 in C2C12 myoblastcellen

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

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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

Uitputting van de Muis Cellen van geënte, humane tumoren verbetert Downstream Analyse van Target Cellen

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

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

Evaluatie van de opname van de Exon geïnduceerd door Splice switch Antisense Oligonucleotides in SMA patiënt fibroblasten

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