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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 (....
1. C2C12 Cell Culture
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.......
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.......
The authors have nothing to disclose.
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).
....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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