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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 (....

Protocol

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 cells. Place the plate in a 37 °C, 5% CO2 incubator for 3 - 5 min.
  3. Neutralize the trypsin by adding 3 ml growth medium. Pipette ....

Representative Results

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.......

Discussion

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.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
DMEM, high glucoseLife Technologies11965-084for culture medium
Fetal Bovine Serum - PremiumAtlanta BiologicalsS11150for culture medium
Penicillin-Streptomycin-Glutamine (100X)Life Technologies10378-016for culture medium
Insulin from bovine pancreasSigma AldrichI6634-100MGfor differentiation medium
equine serumAtlanta BiologicalsS12150for differentiation medium
FuGENE HD Transfection ReagentPromegaE2311 for transfection
G418 sulfate Gold Biotechnology G-418-10for drug resistant selection
Puromycin dihydrochlorideSigma Aldrichsc-108071for drug resistant selection
NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 wellLife TechnologiesNP0323BOXfor western blot
NuPAGE Transfer Buffer (20X)Life TechnologiesNP00061for western blot
NuPAGE MES SDS Running Buffer (20X)Life TechnologiesNP0002for western blot
Amersham Protran Supported 0.2 NC, 300mmx4mGE healthcare life science10600015for western blot
MF 20Developmental Hybridoma BankMF 20primary Ab for immunostaining
Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugateThermo Fisher ScientificT-862secondary Ab for immunostaining
One step qRT-PCR MasterMixAnaSpec05-QPRT-032Xfor qRT-PCR
TriPure Isolation ReagentRoche11667165001for RNA isolation
CUG-BP1 Antibody (3B1)santa cruzsc-20003primary Ab western blot
Actin Antibodysanta cruzsc-1615goat polyclonal IgG for loading control
293T EcopackClontech631507cells for retrovirus preparation
pMSCV-puroClontech634401empty retroviral vector for retrovirus preparation
pMSCV-Celf1Flag-purohouse-constructednot availableretroviral vector encoding Celf1Flag, used in retrovirus preparation
psPAX2gift from Didier Trononot availablefor lentivirus preparation
pMD2.Ggift from Didier Trononot availablefor lentivirus preparation
GFP-CUG5gift from M.S. Mahadevannot availabledetails in reference 10 
GFP- CUG200gift from M.S. Mahadevannot availabledetails in reference 10 
Triton X-100Sigma AldrichX100for immunostaining
paraformaldehydeSigma AldrichP6148for immunostaining
TWEEN 20Sigma AldrichP9416for immunostaining
DAPISigma AldrichD9542for immunostaining

References

  1. Harper, P. S. . Myotonic dystrophy. 3rd edn. , (2001).
  2. Timchenko, L. Molecular mechanisms of muscle atrophy in myotonic dystrophies. Int J Biochem Cell Biol. 45 (10), 2280-2287 (2013).
  3. Brook, J. D., et al.

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