RNAi Technology to Study In Vivo Muscle Metabolism

描述（由申请人提供）：骨骼肌是体内最大的葡萄糖储存库，胰岛素促进肌肉葡萄糖摄取的作用缺陷可导致循环葡萄糖浓度升高和肌糖原浓度改变。尽管了解骨骼肌中胰岛素作用的分子机制很重要，主要是由于技术原因，但对该组织的关注相对较少。RNA干扰（RNAi）技术是研究特定基因表达的有力工具，但由于基因传递系统的限制，其最近在哺乳动物中的应用仍主要局限于细胞模型。我们最近的研究表明，电穿孔是一种在体内有效转染骨骼肌的可靠方法，从而为在骨骼肌中实施RNAi技术开辟了道路。然而，为了使这项技术适应骨骼肌，我们需要对方法进行重大调整。该试点和可行性项目的具体目标是：1)开发一种使用RNAi基因沉默进行成人骨骼肌体内研究的方法；2)确定RNAi基因沉默方法是否可用于破坏体内骨骼肌中的葡萄糖转运和胰岛素作用；3)利用RNAi基因沉默方法改善骨骼肌胰岛素抵抗。RNAi方法在骨骼肌中的成功实施将为研究体内调节肌肉代谢和功能的分子机制以及阐明骨骼肌胰岛素抵抗的具体机制提供极有价值的工具。最终，这种方法可以为骨骼肌胰岛素抵抗和其他慢性肌肉疾病（如营养不良）的治疗提供基础。

DESCRIPTION (provided by applicant): Skeletal muscle is the largest storage reservoir for glucose in the body, and defects in insulin action to promote muscle glucose uptake can result in elevations of circulating glucose concentrations and altered muscle glycogen concentrations. Despite the importance of understanding the molecular mechanisms of insulin action in skeletal muscle, primarily for technical reasons, there has been relatively little focus on this tissue. RNA interference (RNAi) technology for knocking-down the expression of specific genes is a powerful tool to study gene function, but its recent application to mammals is still largely restricted to cell models due to limitations in gene delivery systems. Our recent studies demonstrate that electroporation is a reliable method for efficient transfection of skeletal muscle in vivo, and thus opens the way for implementing the RNAi technology in skeletal muscle. However, for this technique to be adapted to skeletal muscle we will need to make significant adjustments to the methodology. The specific aims for this pilot and feasibility project are: #1) To develop a method to use RNAi gene silencing for studies of adult skeletal muscle in vivo; #2) To determine if the RNAi gene silencing approach can be used to disrupt glucose transport and insulin action in skeletal muscle in vivo; and #3) To use the RNAi gene silencing approach to ameliorate skeletal muscle insulin resistance. The successful implementation of RNAi methodology in skeletal muscle will provide an extremely valuable tool to study the molecular mechanisms regulating muscle metabolism and function in vivo, as well as for elucidating specific mechanisms underlying skeletal muscle insulin resistance. Ultimately, this approach could provide the basis for treatment of skeletal muscle insulin resistance and other chronic muscle diseases such as the dystrophies.