title: Protein overexpression in skeletal muscle using plasmid-based gene transfer to elucidate mechanisms controlling fiber size.
abstract: Plasmid DNA electrotransfer is a direct method of gene delivery to skeletal muscle commonly used to identify endogenous signaling pathways that mediate muscle remodeling or pathological states in adult rodents. When plasmids encoding a protein to be overexpressed are fused to a fluorescent protein or an epitope-tag, plasmid electrotransfer permits visualization of the expressed protein in muscle fibers. Here, we demonstrate the use of electrotransfer of plasmids encoding mutant or wild type proteins to identify the role of the endogenous protein in regulating muscle fiber atrophy. The plasmids used encode a dominant negative form of the inhibitor of kappaB kinase beta (IKKβ) fused to green fluorescent protein (GFP), a constitutively active form of IKKα fused to GFP, and a wild type IKKβ fused to an HA tag. We show the effects of overexpression of these proteins on rat or mouse fiber size either with disuse atrophy or in normal weight bearing muscle. The effects of overexpressed proteins on myofiber size are assessed by comparing cross-sectional area of the transfected, fluorescent myofibers to the nontransfected, nonfluorescent myofibers. Using optimized intramuscular plasmid DNA injection and electroporation, we illustrate high transfection efficiency with no overt muscle damage using medium sized fusion proteins (105 kDa).
authors: Department of Health Sciences, Boston University, Boston, MA, USA.
authors: Chia-Ling
authors: CL
authors: Wu
authors: Susan C
authors: SC
authors: Kandarian
mesh_terms: D000818:Animals; D018274:Electroporation / Q000379:methods; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D007273:Injections, Intramuscular; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats
chemical_list: D049452:Green Fluorescent Proteins; D051550:I-kappa B Kinase