Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 (MBNL1), CUGBP, and Elav-like family 1 (Celf1), are misregulated. Due to the sequestration on the expanded CUG repeats, MBNL1 loses its ability to regulate alternative splicing⁵. Celf1, on the other hand, is up-regulated^6,7. Overexpression of Celf1 is associated with muscle loss and weakness, which are not attributed to loss of MBNL1 function. Animal models simulating DM1-related alterations, including DMPK 3'-UTR CUG expansion, loss of MBNL1, and overexpression of Celf1, have been established. However, modeling DM1 in myoblasts offers an efficient alternative, especially for dissecting DM1-related cellular and molecular events.

C2C12 myoblast cell line was first isolated from injured C3H mouse muscle and widely used to study myogenic differentiation^8,9. C2C12 cells rapidly proliferate in fetal bovine serum (FBS)-containing media and readily undergo differentiation when FBS is depleted. Yet, using this myoblast differentiation model presents two challenges: C2C12 cells are often resistant to gene transfection/viral transduction; and slight variations in cell handling and differentiation procedure can lead to marked changes in myotube formation.

Our lab routinely uses C2C12 myoblasts as a cell model and has developed protocols that effectively deliver genes by plasmid transfection, retroviral transduction, and lentiviral transduction into C2C12 cell line¹⁰. In the video, we demonstrate the optimized procedures for transfecting/transducing C2C12 cells and maintaining differentiation consistency in establishing DM1 myoblast models.

Access restricted. Please log in or start a trial to view this content.

Protokół

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 6 - 8 times to suspend the cells. Add 1 ml of suspended cell to a new 100 mm plate and incubate at 37 °C, 5% CO₂.

2. C2C12 Cell Transfection/transduction and Selection

1. C2C12 plasmid transfection
   1. 24 hr prior to transfection, plate 7.0 x 10⁵ C2C12 cells into one well of a six-well plate for each transfection.
      Note: This leads to 50 - 60% confluence by the time of transfection.
   2. Allow the transfection reagent to come to room temperature prior to use.
   3. Add 2 µg of plasmid DNA (GFP-CUG5 or GFP-CUG200) into 200 µl pre-warmed serum free medium and vortex briefly. Add 6 µl transfection reagent to the medium and mix immediately for 30 sec using a vortex. Incubate the mixture for 30 min at room temperature.
   4. Change the media of C2C12 cells to 2.5 ml serum-free growth media. Add the transfection mixture to the cells after incubation. Return the plates to a 5% CO₂, 37 °C incubator.
   5. Keep the cells in transfection mixture/serum-free growth media for 4 hr or up to overnight. Change media back to the growth media the next day or when cytotoxicity is apparent.
      Note: Cytotoxicity is assessed by microscopic inspection of the cells. Observe the changes in cell morphology and cell detachment. As long as there is no overt cytotoxicity, overnight incubation in serum-free growth media enhances transfection.
   6. Select the cells 48 hr after transfection by adding 1.2 - 1.6 mg/ml G418 for ~ 10 days until the control culture (transfected without the plasmids) does not have live cells. Change the media every two days with growth media supplemented with G418.
   7. Maintain the G418 resistant cells in growth medium and 5% CO₂ at 37 °C for subsequent experiments.
2. Retrovirus preparation and C2C12 retroviral transduction
   1. Prepare HEK 293 culture medium by supplementing DMEM high glucose with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine.
      1. Approximately 24 hr prior to transfection, plate 4.0 x 10⁶ ecotropic HEK 293-based packaging cells in a 100 mm plate (80% confluence at transfection) containing cell culture medium.
   2. Allow the transfection reagent to come to room temperature prior to use.
   3. Mix 15 µg plasmid DNA (pMSCV-Puro or pMSCV-Celf1Flag-Puro) in 1 ml serum-free growth medium. Vortex briefly. Add 30 µl transfection reagent from step 2.2.2 into the DNA/serum-free growth medium tube, and mix well using vortex. Incubate the mixture at room temperature for 30 min.
   4. Add transfection mixture dropwise to the ecotropic HEK 293-based packaging cells. Gently swirl the plates and return them back to the 5% CO₂, 37 °C incubator. After 24 hr, change the media to 10 ml pre-warmed fresh growth medium.
   5. Harvest the supernatant (containing retrovirus) 48 hr after transfection using a pipette and transfer the supernatant into a 50 ml centrifuge tube. Add 10 ml of new warm media to the plate and return it to the incubator. Keep the supernatant at 4 °C.
      Note: The media is added gently to the side of the well so as not to disrupt the cell monolayer.
   6. Harvest the second batch of supernatant 60 hr after transfection and pool it with the supernatant from 2.2.5. Centrifuge at 500 x g, 4 °C for 7 min to remove cellular debris.
   7. Transfer the supernatant into a new 50 ml centrifuge tube. Use retrovirus to transduce C2C12 cells at this point by proceeding to step 2.2.9 or aliquot and store the supernatant at -20 °C for future use.
   8. Thaw the retrovirus from 2.2.7 at room temperature, if a frozen stock is used here.
      Note: Avoid multiple freeze-thaw cycles.
   9. Plate C2C12 cells on the same day of transduction aiming for 30 - 40% of confluence.
      1. Discard the growth medium and wash C2C12 cells with 3 ml room temperature PBS. Remove the PBS, add 500 µl 0.25% Trypsin-EDTA, and incubate the plate in a 37 °C, 5% CO₂ incubator for 3 - 5 min.
      2. Neutralize the trypsin by adding 3 ml growth medium. Pipette 6 - 8 times to suspend the cells. Count cells using a hemocytometer and add 8.0 x 10⁵ C2C12 cells to a 60 mm plate.
   10. Add 0.5 ml retrovirus to infect one 60 mm plate of cells in 3 ml growth media. Return the plate to the incubator.
   11. Replace with 3 ml fresh media supplemented with selection antibiotic (puromycin 1 - 3 µg/ml) 48 hr after infection. Replace 3 ml medium with antibiotic every day for ~ 5 days until the untransduced C2C12 control culture dies out.
3. Lentivirus preparation and C2C12 lentiviral transduction
   Note: Perform all the lentivirus-related procedures in Biosafety Level 2 cabinet and follow biological safety guidelines. Liquid waste containing the virus must be disinfected prior to disposal.
   1. Plate 4.0 x 10⁶ 293T cells in a 100 mm plate (~ 80% confluence at day of transfection) with HEK 293 cell culture medium (DMEM high glucose, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine) 24 hr before transfection.
   2. Warm the transfection reagent to room temperature. Mix the lentiviral packaging vectors (4 µg pMD2.G and 6 µg psPAX2) together with 8 µg lentiviral transfer vector (Celf1 shRNA or scrambled control shRNA) in 1 ml serum free cell culture medium. Vortex briefly.
   3. Add 36 µl pre-warmed transfection reagent to the DNA/DMEM tube and mix well by vortex. Incubate the mixture at room temperature for 30 min.
   4. Remove the 293T cells from the incubator and add the transfection mixture to the plate. Gently swirl the plate and return it back to 5% CO₂, 37 °C incubator. After 24 hr, replace the transfection mixture with 10 ml fresh culture medium.
   5. Collect the supernatant containing lentivirus 48 hr after transfection and transfer it into a 50 ml centrifuge tube. Add 10 ml new warm media to the plate and return it to incubator. Store the supernatant at 4 °C.
   6. Collect the second batch of supernatant 60 hr after transfection and combine it with the supernatant from 2.3.5. Centrifuge briefly at 500 x g, 4 °C for 7 min.
   7. Transfer the supernatant into a fresh 50 ml centrifuge tube. If using fresh virus, proceed to step 2.3.9. For future use, store virus-containing supernatant at -20 °C in aliquots of 10 ml.
   8. Thaw the virus from 2.3.7 at room temperature before use, if a frozen stock is used.
      Note: Avoid multiple freeze-thaw cycles.
   9. Plate C2C12-CUG200 cells (obtained in Section 2.1) as described in 2.2.9 on the same day of transduction.
      Note: Aim for 30 - 40% of confluence.
   10. Add 0.5 ml virus to infect a 60 mm plate of C2C12-CUG200 and return the plate to the incubator.
   11. Replace the culture medium with fresh media supplemented with the selection antibiotic (puromycin 1 - 3 µg/ml) 72 hr after transduction. Refresh culture media everyday with the selection antibiotic for ~5 days until all untransduced C2C12-CUG200 control culture dies out.
   12. Use Western blot to verify Celf1 knockdown in GFP-CUG200 transfected cells.

3. C2C12 Cell Differentiation

1. Plate 2 x 10⁶ cells in one well of a 6-well plate in 2.5 ml growth media 24 hr before initiation of differentiation.
   Note: The plating density may be adjusted so that full confluence is reached in 24 hr.
   1. Rinse confluent cells with PBS and add 2.5 ml of low-serum differentiation medium (Dulbecco's modified Eagle's medium supplemented with 2% equine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 1 µM insulin). Replace the medium every two days.
      Note: Keep stock insulin frozen and add it to the media at each medium change.
2. During C2C12 differentiation, collect samples for quantitative RT-PCR (see section 4) at the following time points: Day0, 1, 2, 4, and 6. Process the culture for immunostaining at day 6 - 8 (see section 5).

4. RNA Isolation and Quantitative RT-PCR

1. Use the manufacturer's protocol for RNA isolation. After isolation, resuspend the RNA pellet in 30 µl RNase-free water and pipet up and down several times.
   Note: The samples may proceed to Quantitative RT-PCR or can be stored at -80 °C.
2. Use a RT qPCR kit for Quantitative RT-PCR¹⁰ and follow the manufacturer's instruction. Prepare the reaction mix according to Table 1.

Component	Volume (µl)	Final Concentration
2x reaction buffer	10	1x
Forward primer	0.5	200 nM
Reverse primer	0.5	200 nM
Probe	0.5	200 nM
Reverse Transcriptase & RNase Inhibitor	0.1	0.25 U/ml
Template	1	100 ng total RNA
RNase free water	7.4	NA
Total Mix	20

Table 1. Quantitative RT-PCR Master Mix Preparation

3. Run a 20 µl RT-qPCR reaction using thermal profile Table 2.

Reverse transcription step	30 min, 48 °C
DNA polymerase activation/Reverse Transcriptase inactivation	10 min, 95 °C
40 Cycles	15 sec, 95 °C
	1 min, 60 °C

Table 2. Quantitative RT-PCR thermal profile

5. Immunostaining

1. Fix the monolayer culture in 2.5 ml freshly prepared 4% paraformaldehyde/PBS at room temperature for 10 min.
2. Permeabilize the samples in 2.5 ml 0.1% Triton X-100/PBS for 30 min at room temperature.
3. Transfer the samples to a humidified chamber. Block the samples in 2.5 ml blocking buffer (0.1% nonionic surfactant/PBS, 10% normal goat serum) for 30 min at 37 °C.
4. Discard blocking buffer and apply 800 µl primary antibody against Myosin Heavy Chain diluted in blocking buffer (1:100). Incubate in a humidified chamber overnight at room temperature.
5. Wash the plate 3 times (10 min each) with 2.5 ml wash buffer (0.1% polysorbate 20/PBS) on day 2.
6. Remove the wash buffer and apply 800 µl secondary antibody (goat anti-mouse IgG, 1:500 diluted in PBS). Incubate in a humidified chamber at room temperature for 1 hr.
7. Wash two times (10 min each) with 2.5 ml wash buffer.
8. Incubate with 800 µl DAPI (1 µg/ml, diluted in distilled deionized H₂O) for 5 min.
9. Wash with 2.5 ml wash buffer again for 10 min.
10. Change the washing buffer to 2.5 ml PBS and use a fluorescent microscope to capture images.

Access restricted. Please log in or start a trial to view this content.

Wyniki

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...

Access restricted. Please log in or start a trial to view this content.

Dyskusje

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...

Access restricted. Please log in or start a trial to view this content.

Materiały

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, 300 mm x 4 m	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