Macino Martina, Biferali Beatrice, Cipriano Andrea, Ballarino Monica, Mozzetta Chiara
Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR) at Sapienza University of Rome, Rome, Italy.
Department of Biology and Biotechnologies "Charles Darwin", Sapienza University of Rome, Rome, Italy.
Bio Protoc. 2021 Nov 5;11(21):e4209. doi: 10.21769/BioProtoc.4209.
LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics , such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.
长链非编码RNA（lncRNAs）最近被认为参与了肌肉分化的表观遗传调控，其功能表征传统上依赖于肌源性分化模型。然而，使用实验范式特异性靶向肌肉干细胞（MuSCs，也称为卫星细胞）中的lncRNAs表达，是在更生理的背景下探究其功能的重要前提条件。由于分离和培养单个肌纤维可将卫星细胞保留在其周围基膜下方的生理微环境中，该方法是追踪卫星细胞动态变化（如从静止状态激活、定向祖细胞的扩增、分化和自我更新）的最佳途径。在这里，我们详细介绍一种优化方案，用于从成年小鼠的趾长伸肌（EDL）骨骼肌中分离出有活力的单个肌纤维，并通过反义锁核酸GapmeRs介导的敲低（KD）来调控lncRNAs的表达。此外，我们描述了一种EdU掺入方法，该方法与lncRNA KD以及随后对增殖、分化和卫星细胞特异性标记物的免疫荧光分析相结合，能够推断lncRNAs对肌肉干细胞动态变化的功能。图形摘要：实验工作流程展示了方案步骤的主要环节：从小鼠后肢采集EDL肌肉；将EDL消化成单个肌纤维；用反义寡核苷酸转染并培养96小时；免疫荧光方案及图像结果。
