The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme β-galactosyl-3-kinase. The mature muscle cells of mammalian skeletal muscle, known as myofibers, are multinucleated syncytia that arise from the fusion of mononucleated precursors, or myoblasts. Myoblasts persist in mature muscle as satellite cells, continue to fuse to adjacent myofibers during postnatal growth, and provide a source of cells for new muscle formation during muscle regeneration after injury. The first indication that myoblasts could be used for therapeutic purposes was the finding that transplantation of minced muscle from one animal to another resulted in the formation of hybrid myofibers, which are essentially heterokaryons composed of nuclei from both animals. This observation led to the idea that myoblasts, grown in vitro, could be used as "cell therapy" for hereditary muscle diseases. By fusing with mature or regenerating fibers of the host, implanted myoblasts could form hybrid myofibers thus contributing to the syncytium a normal gene product that was missing from host muscle. This principle has been most successfully applied to muscular dystrophies in mice. For example, the mdx mouse, which is the genetic homolog of the human disease Duchenne muscular dystrophy, has a defect in the structural gene, dystrophin. In the mouse, as in humans, the absence of dystrophin leads to focal muscle necrosis with cycles of muscle degeneration and regeneration. Transplantation of normal myoblasts into mdx muscle leads not only to the expression of dystrophin in hybrid fibers, but also to the protection of those fibers from the characteristic pathologic changes. In addition to the treatment of intrinsic muscleThe transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme β-galactosyl-3-kinase. The mature muscle cells of mammalian skeletal muscle, known as myofibers, are multinucleated syncytia that arise from the fusion of mononucleated precursors, or myoblasts. Myoblasts persist in mature muscle as satellite cells, continue to fuse to adjacent myofibers during postnatal growth, and provide a source of cells for new muscle formation during muscle regeneration after injury. The first indication that myoblasts could be used for therapeutic purposes was the finding that transplantation of minced muscle from one animal to another resulted in the formation of hybrid myofibers, which are essentially heterokaryons composed of nuclei from both animals. This observation led to the idea that myoblasts, grown in vitro, could be used as "cell therapy" for hereditary muscle diseases. By fusing with mature or regenerating fibers of the host, implanted myoblasts could form hybrid myofibers thus contributing to the syncytium a normal gene product that was missing from host muscle. This principle has been most successfully applied to muscular dystrophies in mice. For example, the mdx mouse, which is the genetic homolog of the human disease Duchenne muscular dystrophy, has a defect in the structural gene, dystrophin. In the mouse, as in humans, the absence of dystrophin leads to focal muscle necrosis with cycles of muscle degeneration and regeneration. Transplantation of normal myoblasts into mdx muscle leads not only to the expression of dystrophin in hybrid fibers, but also to the protection of those fibers from the characteristic pathologic changes. In addition to the treatment of intrinsic muscle