Phenotypic and immunologic factors affecting plasmid-mediated in vivo gene transfer to rat diaphragm

Little is known about the molecular mechanisms governing adaptive responses of the diaphragm in the setting of lung disease. By permitting the study of regulatory elements and the effects of overexpressing genes of interest, direct in vivo gene transfer to the diaphragm could be used as a tool to address such questions. Therefore, we evaluated parameters affecting transfection efficiency and duration of foreign gene expression in the diaphragm after plasmid-mediated gene transfer. Reporter gene constructs were injected into adult rat diaphragm and hindlimb muscles. Transfection efficiency at 8-10 days postinjection was decreased in large caliber ( > 1,000 microns2) and type II myosin heavy chain (MHC)-expressing fibers. There were also strong trends toward augmented transfection efficiency in type I MHC- and embryonic MHC-expressing fibers. All diaphragms demonstrated evidence of muscle injury and inflammatory cell infiltrates at this early time point. By 30 days postinjection, however, neither inflammation nor reporter gene expression was detectable in diaphragm or hindlimb muscles of immunocompetent animals. By contrast, immunosuppressed rats (given cyclosporine; 15 mg.kg-1. day-1) showed high levels of foreign gene expression at 30 days postinjection, which remained stable up to 60 days. Therefore, exploitation of plasmid-mediated in vivo gene transfer as a tool for studying regulated gene expression in the diaphragm may be facilitated by the use of immunodeficient animal models.