Transgenic Approach for Investigating MyoD Function and Testicular Injection as a Method for Producing Transgenic Livestock

Abstract

Skeletal muscle, as the primary component of the animal musculoskeletal system, holds significant economic value for land animals in terms of food production. Skeletal muscle satellite cells (SMSCs) play a critical role as myo-specific stem cells in muscle growth and regeneration. These cells have shown great potential in clinical trials for treating Duchenne Muscular Dystrophy and Volumetric Muscle Loss. However, the majority of SMSCs remain in a quiescent state within their muscle fiber niche, making it challenging to harvest a sufficient quantity for clinical applications. In this study, we successfully obtained a large number of goat SMSCs (gSMSCs) with typical characteristics and myo-differentiation ability by modifying the conventional trypsin digestion method. Subsequently, we cloned and modified the MyoD gene, transferring it into goat skin fibroblasts. Remarkably, the MyoD-eGFP-expressing goat skin fibroblasts acquired the ability of myo-differentiation and fused with either each other to form myotubes or fused with the intrinsic myotubes previously formed by gSMSCs. Considering the process of muscle damage regeneration, the incorporation of external fibroblasts in the regeneration process can facilitate the connection of disrupted muscle fibers, thereby accelerating the regeneration time. However, this approach may lead to reduced strength and contractility of the regenerated muscle. Another strategy to expedite muscle regeneration in Volumetric Muscle Loss involves overexpression of MyoD in the damaged muscle or ectopic expression in skin fibroblasts. To further investigate the in vivo biological function of MyoD, transgenic mice and goats were generated via testicular injection, demonstrating a high success rate.