Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

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 ....

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 myogenesis^11-14. 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.......

Materials

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