Development of a clonal equine myoblast cell line capable of terminal differentiation into mature myotubes in vitro
Development of a clonal equine myoblast cell line capable of terminal differentiation into mature myotubes in vitro
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DOI：
10.2460/ajvr.76.7.608
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发表时间：
2015-07-01
影响因子：
1
通讯作者：
Piercy, Richard J.
中科院分区：
文献类型：
--
作者：
Naylor, Rosie J.;Piercy, Richard J.
实验方法为了产生克隆的马成肌细胞系，该细胞系保留在特定条件下分裂多次传代并分化成多核肌管的能力，样品培养来自健康纯种马的原代马骨骼肌衍生细胞。其可由多西环素应答启动子控制。选择稳定整合TAg的细胞并扩增至第25代。对于每次传代，确定细胞的分化和融合特性，并进行免疫细胞化学分析以评估TAg和其他肌肉特异性蛋白的表达。最佳条件下，导致细胞分化成肌管也determined.Prosthesis与未转染的对照细胞相比，肌源性，结蛋白阳性细胞表达的TAg时，在33摄氏度孵育，并可以保持在培养物中的许多代。在37 ℃孵育的细胞中或当与多西环素在33 ℃孵育时，鉴定到TAg表达减少。当细胞与强力霉素在37 ℃孵育时，未检测到TAg的表达，并且当血清从培养基中取出时，这些克隆分化成多核myotubes.CONCLUSION和CLINICAL RELEVANCEResults的纯群体，表明生产永生化克隆马骨骼肌细胞系是可能的。克隆马骨骼肌细胞系将是一个有价值的体外工具，用于马的生理和疾病的研究。
OBJECTIVETo produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions.SAMPLECultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred.PROCEDURESCell 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.RESULTSCompared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33 degrees C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37 degrees C or when incubated with doxycycline at 33 degrees C. Expression of TAg was not detected when cells were incubated with doxycycline at 37 degrees C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes.CONCLUSIONS AND CLINICAL RELEVANCEResults indicated that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.