Hain Brian A, Waning David L
Department of Cellular and Molecular Physiology, Penn State College of Medicine;
Department of Cellular and Molecular Physiology, Penn State College of Medicine.
J Vis Exp. 2022 Apr 6(182). doi: 10.3791/63916.
Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.
通过质粒电穿孔对小鼠骨骼肌进行瞬时基因表达调控是评估正常和病理生理学的有用工具。靶基因的过表达或敲低使研究人员能够操纵个体分子事件，从而更好地理解影响肌肉质量、肌肉代谢和收缩性的机制。此外，编码荧光标签的DNA质粒的电穿孔使研究人员能够在体内测量骨骼肌中蛋白质亚细胞定位的变化。骨骼肌的一项关键功能评估包括肌肉收缩性的测量。在本方案中，我们证明在质粒DNA注射、电穿孔和基因表达调控后，仍可进行全肌肉收缩性研究。本指导程序的目的是演示将DNA质粒电穿孔到小鼠骨骼肌中的逐步方法，以促进其在胫骨前肌和趾长伸肌的肌纤维中的摄取和表达，并证明注射和电穿孔不会损害骨骼肌的收缩性。
