Development of a clonal equine myoblast cell line capable of terminal differentiation into mature myotubes in vitro.
Abstract: To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. Methods: Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. Methods: Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined. Results: Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes. Conclusions: Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.
Publication Date: 2015-06-26 PubMed ID: 26111090DOI: 10.2460/ajvr.76.7.608Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research describes the creation of a clonally reproducible equine myoblast cell line that can multiply extensively and mature into myotubes under specific conditions. This development can be a valuable tool in equine physiology and disease research.
Objective and Methodology
- The primary goal of this research was to develop a clonal equine myoblast cell line that can both divide for many passages and morph into multinucleated myotubes under certain conditions.
- The researchers derived primary cells for this research from the skeletal muscle of a healthy Thoroughbred horse and cultured them in a laboratory.
- The cultured cells were then subjected to a process called electroporation using a plasmid (pNIT). This plasmid regulates the expression of the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg).
- The function of the TAg is to allow the cells to grow endlessly, which is crucial for the creation of a lasting cell line.
Procedure, Analysis, and Results
- Following the electroporation process, cells that stably absorbed the TAg were selected and moved through 25 rounds of expansion (growth or reproduction of cells).
- At each of these 25 steps, the differentiation (maturation of cells into a specific type) and fusion properties of the cells were ascertained.
- Furthermore, the researchers conducted immunocytochemical analysis to evaluate the expression of TAg and other muscle-related proteins in the cells.
- They discovered that cells with the TAg ‘switched on’ were able to continuously grow. They also identified that ceasing the expression of the TAg via two methods (increasing the incubation temperature or introducing doxycycline) successfully initiated the transformation of the cells into a population of multinucleated myotubes.
- The research successfully produced a clonal, or genetically identical, equine myoblast cell line that maintained the capability to differentiate into mature myotubes.
Conclusion and Implications
- Findings from the study demonstrated the successful creation of an immortalized clonal equine skeletal muscle cell line.
- This cell line offers promising potential as an in vitro tool in researching equine physiology and diseases, opening pathways for further advancements in this field.
Cite This Article
APA
Naylor RJ, Piercy RJ.
(2015).
Development of a clonal equine myoblast cell line capable of terminal differentiation into mature myotubes in vitro.
Am J Vet Res, 76(7), 608-614.
https://doi.org/10.2460/ajvr.76.7.608 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Cell Differentiation
- Cell Line
- Cells, Cultured
- Horses / physiology
- Male
- Muscle Fibers, Skeletal / cytology
- Muscle Proteins / genetics
- Muscle, Skeletal / cytology
- Myoblasts / cytology
- Stem Cells
- Transfection / veterinary
Citations
This article has been cited 2 times.- Rooney MF, Neto NGB, Monaghan MG, Hill EW, Porter RK. Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.. Biochem Biophys Rep 2023 Mar;33:101391.
- Amilon KR, Cortes-Araya Y, Moore B, Lee S, Lillico S, Breton A, Esteves CL, Donadeu FX. Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells.. Cell Reprogram 2018 Oct;20(5):275-281.
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