Myoblast transfer has been suggested as a method of gene complementation for primary myopathics and to supplement proteins that may be absent in the systemic circulation. This system also offers a unique opportunity to evaluate muscle development and regeneration. It is proposed to study the cell biology of the myoblast transfer system using fetal hosts and stably marked myoblasts. The use of fetal hosts will overcome the problems inherent in myoblast transfer into more mature hosts (i.e., problems of delivery, dispersal and integration of donor myoblasts into a significant number of myofibers and problems of immune rejection), as at the time of injection the small developing muscles of the immune-incompetent fetal hosts will be undergoing extensive de novo myotube formation and will have poorly organized connective tissue. This millieu should result in the production of a significant number of widely dispersed mosaic myotubes (with different frequencies of identifiable donor myoblast), without resorting to the muscle traumatization required to induce myoblast incorporation into muscles containing their full complement of myofibers. This will permit evaluation of the fate of the injected myoblast, determining whether some of their progeny are maintained as myosatellite cell, capable of participating in myofiber growth and regeneration and of the ability of donor-derived myonuclei to survive long-term and to continue to produce a unique structural protein product. The question of myoblast commitment to specific cell lineages will then be addressed by transferring stably marked myoblasts from embryonic muscle and myosatellite cells from adult muscle into developing fetal muscle. We will also address -the potential benefit of using myoblast transfer as a method of gene complementation for the replacement of dystrophin into the dystrophin-deficient myofibers of fetal mdx mice. The frequency of donor to host myonuclei in dystrophin-positive mosaic myofibers in the mdx mice and the effectiveness of dystrophin replacement on modifying the degeneration-regeneration of dystrophin deficient muscle will be assessed. Evaluations will be made with immunohistochemistry, and light microscopy and scanning confocal microscopy, and with Western blotting with antibody against dystrophin and antibodies against myosin or induction. The results of the proposed experiments will provide significant new information about the myoblast transfer system that will be open the way for future use of this system for therapeutic purposes and as a tool for evaluating many aspects of the cell biology of muscle development.
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