Pimentel Mafalda R, Falcone Sestina, Cadot Bruno, Gomes Edgar R
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa.
Myology Research Center, UM76-INSERM U974-CNRS FRE 361, Sorbonne University, UPMC University of Paris 6.
J Vis Exp. 2017 Jan 7(119):55141. doi: 10.3791/55141.
Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation. Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.
骨骼肌由肌纤维组成，肌纤维是哺乳动物体内最大的细胞，也是少数的多核细胞之一。构成肌纤维的复杂且在进化上保守的结构是如何组装的仍在研究之中。它们的大小和生理特征常常限制了操作和成像应用。永生化细胞系的培养被广泛使用，但它只能复制分化的早期步骤。在此，我们描述了一种方法，能够对源自原代小鼠成肌细胞的肌纤维进行简便的基因操作。分化一周后，肌纤维表现出收缩性、排列整齐的肌节和三联体，以及外周细胞核。整个分化过程可以通过实时成像或免疫荧光进行追踪。该系统结合了现有体外和体内实验方法的优点。简便高效转染的可能性以及对所有分化阶段的易获取性拓宽了潜在应用范围。随后，肌纤维不仅可用于解决相关的发育和细胞生物学问题，还可用于重现肌肉疾病表型以用于临床应用。
