MARKER PROTEINS FOR MUSCLE CELL TRANSPLANTATION STUDIES

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Despite intensive research, the molecular defects underlying the muscular dystrophies of human and animal tissues have yet to be identified. It is conceivable, however, that a successful therapy might be developed using a method which circumvents the need for this knowledge. Ongoing work from several laboratories indicates that, on introduction of normal muscle cells into diseased muscle, the normal nuclei are incorporated into the host fibers and their phenotype expressed. However, identification of the hypothesized hybrid cells and correlation with improved fiber structure has not been possible because of the lack of independent markers for normal and dystrophic nuclei. We have identified three nondiseased-related, allelic variants of myosin light chain-1 (types I, II and III) in the fast white muscle fibers of domestic chickens suitable for use as marker proteins. It is proposed: (i) to develop strains of noromal chickens homozygous for the type I and III LC-1 variants (birds of all dystrophic strains are fortuitously homozygous for the type II form). (ii) to develop monoclonal antibodies specific for each of the variants. (iii) to determine, by immunomicroscopy in culture, using two kinds of normal myocytes differing in LC1 types, whether LC1 is limited in diffusion in the myofiber. A positive finding will permit location of normal and dystrophic nuclei within a hybrid fiber in subsequent transplantation experiments. (iv) to determine, using immunomicroscopy and normal and dystrophic cell types differing in LC1 variants, the survival advantages of normal and dystrophic cells mixed in culture. (v) to determine in vivo, using immunomicroscopy, the extent of hybrid fiber formation following introduction of normal cells expressing one LC1 type into dystrophic tissue expressing a second, and to correlate this with any improvement in fiber histology and ultrastructure. (vi) if LC1 diffusion in the fiber proves to be limited, permitting identification of normal and dystrophic nuclei in hybrid fibers (see (iii) above), to compare the fiber histology and ultrastructure in regions of hybrid fibers maintained by normal and dystrophic nuclei respectively. (vii) using the LC1 variants and the normal and dystrophic myosin heavy chains (HC) distinguished by us, to use similar procedures to determine whether LC1 and HC occupy the myofiber to similar extents.