We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.
Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.
Extracellular matrix (ECM) proteins provide structural support for all tissues, mediate cell-cell communication, and determine cell fate. The formation and dynamic remodeling of ECM is thus critical to maintain tissue and organ homeostasis1,2. Pathological variants in several genes coding for ECM proteins give rise to musculoskeletal disorders with phenotypes ranging from muscular dystrophies to pseudomouscular build3,4. For example, pathogenic variants in ADAMTSL2 cause the extremely rare musculoskeletal disorder geleophysic dysplasia, which presents with pseudomuscular build, i.e., an apparent increase in skeletal muscle mass5. Together with gene expression data in mouse and humans, this suggests a role for ADAMTSL2 in skeletal muscle development or homeostasis6,7.
The protocol that we describe here was developed to study the mechanism by which ADAMTSL2 modulates skeletal muscle development and/or homeostasis in a cell culture setting. We stably knocked down ADAMTSL2 in the murine C2C12 myoblast cell line. C2C12 myoblasts and their differentiation into myotubes is a well-described and widely used cell culture model for skeletal muscle differentiation and skeletal muscle bioengineering8,9. C2C12 cells go through distinct differentiation steps after serum withdrawal, resulting in the formation of multinucleated myotubes after 3−10 days in culture. These differentiation steps can be reliably monitored by measuring mRNA levels of distinct marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC). One advantage of generating stable gene knockdowns in C2C12 cells is that genes that are expressed at later stages of C2C12 differentiation can be targeted more efficiently, compared to transient knockdown achieved by small-interfering (si) RNA, which typically lasts for 5−7 days after transfection, and is influenced by the transfection efficiency. A second advantage of the protocol as described here is the relatively fast generation of batches of C2C12 knockdown cells using puromycin selection. Alternatives, such as CRISPR/Cas9-mediated gene knockout or the isolation of primary skeletal muscle cell precursors from human or target-gene deficient mice are technically more challenging or require the availability of patient muscle biopsies or target-gene deficient mice, respectively. However, similar to other cell culture based approaches, there are limitations in the use of C2C12 cells as model for skeletal muscle cell differentiation, such as the two-dimensional (2D) nature of the cell culture set-up and the lack of the in vivo microenvironment that is critical to maintain undifferentiated skeletal muscle precursor cells10.
1. Preparing the shRNA Plasmid DNA from Escherichia coli
2. Culturing and Transfection of C2C12 Cells and Puromycin Selection
3. Phenotypic Analysis of C2C12 Differentiation
NOTE: The methods described below can easily be adapted for general phenotypic analysis of C2C12 myoblast differentiation into myotubes by varying the specific antibodies used in Western blotting or the gene specific primers used in the quantitative polymerase chain reaction (qPCR) analysis.
Selection of puromycin-resistant C2C12 can be achieved in 10−14 days after transfection due to efficient elimination of non-resistant, i.e., untransfected cells (Figure 1B). Typically, more than 80% of the cells detach from the cell culture dish and these cells are removed during routine cell maintenance. Puromycin-resistant C2C12 cells expressing the control (scrambled) shRNA retain the spindle-shape, elongated cell morphology at low cell density and the capability to differentiate into myotubes. C2C12 differentiation upon serum withdrawal can be monitored by bright field microscopy and by immunostaining for the myotube marker myosin heavy chain (MyHC) (Figure 2). MyHC-positive myotubes are observed between 3−5 days after differentiation initiation. Myotubes are multinucleated as shown by the presence of more than one DAPI-positive nucleus within the MyHC-positive cell boundaries. Figure 3A shows bright field images of stable C2C12 cells cultured in complete DMEM. The knockdown efficiency presented here ranges from 40−60% (Figure 3B). Since the mRNA was harvested in the proliferative state where little Adamtsl2 is expressed, the knockdown efficiency appears low, but the knockdown efficiency is expected to be larger at later time points during C2C12 differentiation, where endogenous Adamtsl2 is induced and thus expressed at much higher levels. Western blot analysis confirmed the successful knockdown of ADAMTSL2 in the cell lysate obtained from C2C12 cells stably expressing shRNA 3086 compared to control shRNA (Figure 3C).
Figure 1: Selection of stable C2C12 cells after transfection with shRNA-encoding plasmid DNA. (A) Table showing the target region (CDS, coding sequence; 3'-UTR, 3'-untranslated region), clone ID (hereafter referred to as 1977, 3086, and 972), and sequence of the shRNAs used to target Adamtsl2. (B) The panels show the selection of C2C12 cells transfected with the control shRNA and the three Adamtsl2-targeting shRNAs. C2C12 cells were transfected with the shRNA plasmids and puromycin was added to the medium after 24 h. Puromycin-sensitive cells appear round and eventually detach during routine cell culture maintenance (red arrows). In contrast, puromycin-resistant cells harboring the integrated shRNA plasmids appear spindle-shaped, slightly elongated, attached, and viable (blue arrows). Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 2: C2C12 myoblast to myotube differentiation. Bright field images of differentiating C2C12 cells show dense cobblestone appearance of the cells at the beginning of the differentiation (day 0−1) and multinucleated myotubes were observed after day 5 (upper row). Immunostaining of differentiating C2C12 cells with myosin heavy chain (MyHC, red), which is a marker for myotubes, is induced at day 3 of differentiation (middle panel). Nuclei were stained with DAPI and the merged image is shown in the lower panels. Scale bars = 50 µm. Please click here to view a larger version of this figure.
Figure 3: Validation of stable knockdown in proliferating C2C12 cells. (A) Bright field images of stable C2C12 cells cultured in complete DMEM. Scale bars = 300 µm. (B) qRT-PCR analysis of Adamtsl2 mRNA expression in stable C2C12 cells. Ct values were normalized to the housekeeping gene Hprt1. mRNA was harvested before the onset of differentiation. Error bars represent standard deviation. (C) Western blot analysis showing reduced ADAMTSL2 protein in the cell lysate/ECM fraction from C2C12 cells stably expressing shRNA 3086. Endogenous ADAMTSL2 was detected using a custom-made polyclonal peptide antibody (available upon request). Please click here to view a larger version of this figure.
We describe here a protocol for the stable knockdown of ECM proteins in C2C12 myoblasts and for phenotypic analysis of the differentiation of C2C12 myoblasts into myotubes. Several factors determine the outcome of the experiment and need to be considered carefully. Maintaining C2C12 cells in the proliferating phase is a critical step to keep the C2C12 cells in the myoblast precursor state. Retaining the capability of C2C12 cells to consistently differentiate into myotubes depends on i) the passage number of the cells, ii) the density of the cultured cells during routine maintenance, and iii) nutrient availability, requiring frequent and regular replenishment of the cell culture medium11,12,13. Due to some unknown mechanisms, higher passage number C2C12 cells also lose the potential for further myoblast fusion11. The instructions from the provider of the C2C12 cells suggest maintaining these cells up to passage number 15. Thereafter, the differentiation potential may be reduced and experiments with such cells may result in less consistent myotube formation. On the other hand, the cell density during maintenance can result in similar effects9. Reaching confluent C2C12 cell densities during routine cell culture promote initiation of C2C12 myoblast differentiation and thus may negatively influence the differentiation potential of the cell population. Therefore, it is of critical importance to prevent C2C12 cells from reaching high cell densities during routine C2C12 cell maintenance. This can be achieved by already sub-culturing C2C12 cells at low cell densities (<50−60% confluence). Serum starvation is used to induce C2C12 cell differentiation into myotubes. Therefore, maintaining cells for longer times without replenishing medium severely exhaust the nutrient and serum levels. Replenishing with fresh serum containing medium at least every two days can prevent the onset of unwanted premature differentiation due to nutrient and serum deprivation.
C2C12 differentiation is typically induced by serum starvation. The percentage of serum used to induce C2C12 differentiation can greatly influence the results, specifically the time it takes to form MyHC positive myotubes14,15. Several protocols show successful induction of differentiation under various serum concentrations. Use of 2−10% FBS or horse serum and complete serum deprivation has been reported and all conditions result in C2C12 myotube formation. The serum percentage or change in the serum lot can significantly alter markers for differentiation. In addition, the source of the serum may affect the experimental outcome, because the country of origin may or may not allow certain additives during bovine serum production8. The serum level could be adjusted to achieve the differentiation rate according to specific experimental requirements. Insulin can be added to the culture medium of C2C12 cells to accelerate differentiation and myotube formation16.
The ability to deliver plasmids encoding shRNA or recombinant proteins into C2C12 cells via transfection is an attractive feature of C2C12 cells. Several commercial liposome-based transfection reagents have been used previously to deliver plasmid DNA into C2C12 cells with variable reported transfection efficiencies17,18,19,20,21. PEI also shows reasonable transfection efficiency and is a cost-effective alternative to liposome-based transfection reagents. A comparison between transfection with a commercial liposome-based transfection reagent and PEI showed no considerable change in the transfection efficiency and slightly higher efficiency was found with PEI22. In our hands, transfection efficiency with PEI was sufficient to generate stable puromycin-resistant C2C12 cells. However, a critical step in this protocol is to keep the incubation time of the transfection reagent short. As mentioned above, C2C12 cells are sensitive to serum withdrawal and prolonged exposure to low serum conditions during transfection may favor C2C12 cell differentiation. Since transfection is performed in the absence of serum, the incubation time after transfection was kept short to resupply quickly serum-containing medium to prevent premature differentiation. Puromycin was added to the culture medium 24 h after transfection to initiate the selection of puromycin-resistant C2C12 cells. Methods for introducing plasmid DNA into differentiated myotubes include transfection (up to 85% efficiency reported), electroporation, or a biolistic approach23,24,25. Alternatively, stable C2C12 cells harboring an inducible shRNA plasmid could be generated to knock down genes at later stages of myotube differentiation or during myotube maturation26.
The method described here resulted in stable knockdown of ADAMTSL2 in C2C12 cells where its function during differentiation into myotubes can now be determined. The generation of stable knockdown cells may be especially important to study the function of proteins that are induced during myotube formation or maturation. Transient transfection with siRNA for example would not be sufficient to efficiently knockdown these genes, since the transient knockdown effect of siRNA may wean off after 5−7 days. Alternatively, the knockout of an ECM protein using CRISPR/Cas9 is technically more challenging and individual clones need to be selected and sequenced to ensure knockout of the desired gene. However, the evolving line of reagents may render CRISPR/Cas9 the method of choice for loss-of-function experiments in the future.
The authors have nothing to disclose.
D.H. is supported by the National Institutes of Health (National Institute for Arthritis and Musculoskeletal and Skin Diseases, NIAMS, grant number AR070748) and seed funding from the Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mt. Sinai.
Acetone | Fisher Chemical | 191784 | |
Agar | Fisher Bioreagents | BP1423 | |
Ampicillin | Fisher Bioreagents | BP1760-5 | |
Automated cell counter Countesse II | Invitrogen | A27977 | |
Bradford Reagent | Thermo Scientific | P4205987 | |
C2C12 cells | ATCC | CRL-1772 | |
Chamber slides | Invitrogen | C10283 | |
Chloroform | Fisher Chemical | 183172 | |
DMEM | GIBCO | 11965-092 | |
DMSO | Fisher Bioreagents | BP231-100 | |
DNase I (Amplification Grade) | Invitrogen | 18068015 | |
Fetal bovine serum | VWR | 97068-085 | |
GAPDH | EMD Millipore | MAB374 | |
Glycine | VWR Life Sciences | 19C2656013 | |
Goat-anti-mouse secondary antibody (IRDYE 800CW) | Li-Cor | C90130-02 | |
Goat-anti mouse secondary antibody (Rhodamine-red) | Jackson Immune Research | 133389 | |
HCl | Fisher Chemical | A144S | |
Incubator (Shaker) | Denville Scientific Corporation | 1704N205BC105 | |
Mercaptoethanol | Amresco, VWR Life Sciences | 2707C122 | |
Midiprep plasmid extraction kit | Qiagen | 12643 | |
Myosin 4 (myosin heavy chain) | Invitrogen | 14-6503-82 | |
Mounting medium | Invitrogen | 2086310 | |
NaCl | VWR Life Sciences | 241 | |
non-ionic surfactant/detergent | VWR Life Sciences | 18D1856500 | |
Paraformaldehyde | MP | 199983 | |
PBS | Fisher Bioreagents | BP399-4 | |
PEI | Polysciences | 23966-1 | |
Penicillin/streptomycin antibiotics | GIBCO | 15140-122 | |
Petridishes | Corning | 353003 | |
Polypropylene tubes | Fisherbrand | 149569C | |
Protease inhibitor cocktail tablets | Roche | 33576300 | |
Puromycin | Fisher Scientific | BP2956100 | |
PCR (Real Time) | Applied Biosystems | 4359284 | |
Reaction tubes | Eppendorf | 22364111 | |
Reverse Transcription Master Mix | Applied Biosystems | 4368814 | |
RIPA buffer | Thermo Scientific | TK274910 | |
sh control plasmid | Sigma-Aldrich | 07201820MN | |
sh 3086 plasmid | Sigma-Aldrich | TRCN0000092578 | |
sh 972 plasmid | Sigma-Aldrich | TRCN0000092579 | |
sh 1977 plasmid | Sigma-Aldrich | TRCN0000092582 | |
Spectrophotometer (Nanodrop) | Thermo Scientific | NanoDrop One C | |
SYBR Green Reagent Master Mix | Applied Biosystems | 743566 | |
Trichloroacetic acid | Acros Organics | 30145369 | |
Trizol reagent | Ambion | 254707 | |
Trypan blue | GIBCO | 15250-061 | |
Tryptone | Fisher Bioreagents | BP1421 | |
Trypsin EDTA 0.25% | Gibco-Life Technology Corporation | 2085459 | |
Water (DEPC treated and nuclease free) | Fisher Bioreagents | 186163 | |
Western blotting apparatus | Biorad | Mini Protean Tetra Cell | |
Yeast extract | Fisher Bioreagents | BP1422 |