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Protocol

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Medicine

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Published: July 29th, 2016

DOI:

10.3791/54078

1Department of Biology and Biochemistry, University of Houston, 2Department of Economics, University of Houston, 3Department of Cardiology, The First Affiliated Hospital of Nanchang University
* These authors contributed equally

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 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.

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' 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 (....

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1. C2C12 Cell Culture

  1. Maintain C2C12 mouse myoblasts in a 100 mm plate in growth medium (Dulbecco's modified Eagle's medium (DMEM)) supplemented with 20% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Allow C2C12 passaged cells to become approximately 50 - 60% confluent.
  2. Discard the growth medium and wash C2C12 cells with 3 ml room temperature phosphate-buffered saline (PBS). Remove the PBS and add 500 µl 0.25% Trypsin-EDTA to detach the cell.......

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

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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'-UTR, and shRNA into C2C12 cells. The critical points for transfecti.......

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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).

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Name Company Catalog Number Comments
DMEM, high glucose Life Technologies 11965-084 for culture medium
Fetal Bovine Serum - Premium Atlanta Biologicals S11150 for culture medium
Penicillin-Streptomycin-Glutamine (100X) Life Technologies 10378-016 for culture medium
Insulin from bovine pancreas Sigma Aldrich I6634-100MG for differentiation medium
equine serum Atlanta Biologicals S12150 for differentiation medium
FuGENE HD Transfection Reagent Promega E2311  for transfection
G418 sulfate  Gold Biotechnology  G-418-10 for drug resistant selection
Puromycin dihydrochloride Sigma Aldrich sc-108071 for drug resistant selection
NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well Life Technologies NP0323BOX for western blot
NuPAGE Transfer Buffer (20X) Life Technologies NP00061 for western blot
NuPAGE MES SDS Running Buffer (20X) Life Technologies NP0002 for western blot
Amersham Protran Supported 0.2 NC, 300mmx4m GE healthcare life science 10600015 for western blot
MF 20 Developmental Hybridoma Bank MF 20 primary Ab for immunostaining
Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate Thermo Fisher Scientific T-862 secondary Ab for immunostaining
One step qRT-PCR MasterMix AnaSpec 05-QPRT-032X for qRT-PCR
TriPure Isolation Reagent Roche 11667165001 for RNA isolation
CUG-BP1 Antibody (3B1) santa cruz sc-20003 primary Ab western blot
Actin Antibody santa cruz sc-1615 goat polyclonal IgG for loading control
293T Ecopack Clontech 631507 cells for retrovirus preparation
pMSCV-puro Clontech 634401 empty retroviral vector for retrovirus preparation
pMSCV-Celf1Flag-puro house-constructed not available retroviral vector encoding Celf1Flag, used in retrovirus preparation
psPAX2 gift from Didier Trono not available for lentivirus preparation
pMD2.G gift from Didier Trono not available for lentivirus preparation
GFP-CUG5 gift from M.S. Mahadevan not available details in reference 10 
GFP- CUG200 gift from M.S. Mahadevan not available details in reference 10 
Triton X-100 Sigma Aldrich X100 for immunostaining
paraformaldehyde Sigma Aldrich P6148 for immunostaining
TWEEN 20 Sigma Aldrich P9416 for immunostaining
DAPI Sigma Aldrich D9542 for immunostaining

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