title: Optimizing plasmid-based gene transfer for investigating skeletal muscle structure and function.
abstract: Intramuscular injection of naked plasmid DNA is a less cytotoxic alternative to viral vectors for delivering genetic material to skeletal muscle in vivo. However, the low efficiency of plasmid-based gene transfer limits its potential therapeutic efficacy and/or its use for many experimental applications. Current strategies to enhance transfection efficiency (i.e., electroporation) can cause significant muscle damage, confounding physiological assessments such as muscle contractility. Optimizing protocols to limit damage is critical for accurate physiological, biochemical, and molecular measurements. Following extensive testing, we developed an electroporation protocol that enhances transfection efficiency in skeletal muscles without causing muscle damage. Pretreating mouse tibialis anterior muscles with hyaluronidase and electroporation at 75 V/cm (using 50% vol/vol saline as a vehicle for plasmid DNA) resulted in 22 +/- 5% of the muscle fibers expressing a reporter gene. This protocol did not compromise contractile function of skeletal muscles assessed at both the intact (whole) muscle and the cellular (single fiber) level. Furthermore, ectopic expression of insulin-like growth factor I to levels that induced muscle fiber hypertrophy without causing tissue damage or compromising muscle function highlights the therapeutic potential of these methods for myopathies, muscle wasting disorders, and other pathophysiologic conditions.
authors.affiliation: Department of Physiology, The University of Melbourne, Melbourne, VIC 3010, Australia.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Schertzer
authors.forename: David R
authors.initials: DR
authors.lastname: Plant
authors.forename: Gordon S
authors.initials: GS
authors.lastname: Lynch
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics*; D018274:Electroporation; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D007150:Immunohistochemistry; D007273:Injections, Intramuscular; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000473:pathology; D018482:Muscle, Skeletal / Q000378:metabolism*; D010957:Plasmids / Q000008:administration & dosage*
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D004247:DNA; D006821:Hyaluronoglucosaminidase; D016650:Fluorescein-5-isothiocyanate