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).
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Wolff JA, Malone RW, Williams P et al (1990) Direct gene transfer into mouse muscle in vivo. Science 247:1465–1468
Bodine SC, Stitt TN, Gonzalez M et al (2001) Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3:1014–1019
Sartori R, Milan G, Patron M et al (2009) Smad2 and 3 transcription factors control muscle mass in adulthood. Am J Physiol Cell Physiol 296:C1248–1257
Judge AR, Koncarevic A, Hunter RB et al (2007) Role for I{kappa}B{alpha}, but not c-Rel, in skeletal muscle atrophy. Am J Physiol Cell Physiol 292:C372–382
Van Gammeren D, Damrauer JS, Jackman RW et al (2009) The IkappaB kinases IKKalpha and IKKbeta are necessary and sufficient for skeletal muscle atrophy. Faseb J 23:362–370
Senf SM, Dodd SL, McClung JM et al (2008) Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy. Faseb J 22:3836–3845
Kessler PD, Podsakoff GM, Chen X et al (1996) Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci USA 93:14082–14087
Raz E, Watanabe A, Baird SM et al (1993) Systemic immunological effects of cytokine genes injected into skeletal muscle. Proc Natl Acad Sci USA 90:4523–4527
Rizzuto G, Cappelletti M, Maione D et al (1999) Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA 96:6417–6422
Yin D, Tang JG (2001) Gene therapy for streptozotocin-induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo. FEBS Lett 495:16–20
Mitchell-Felton H, Hunter RB, Stevenson EJ et al (2000) Identification of weight-bearing-responsive elements in the skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) gene. J Biol Chem 275:23005–23011
Esser K, Nelson T, Lupa-Kimball V et al (1999) The CACC box and myocyte enhancer factor-2 sites within the myosin light chain 2 slow promoter cooperate in regulating nerve-specific transcription in skeletal muscle. J Biol Chem 274:12095–12102
Kitsis RN, Leinwand LA (1992) Discordance between gene regulation in vitro and in vivo. GeneExpr 2:313–318
Hunter RB, Stevenson E, Koncarevic A et al (2002) Activation of an alternative NF-kappaB pathway in skeletal muscle during disuse atrophy. Faseb J 16:529–538
Hunter RB, Kandarian SC (2004) Disruption of either the Nfkb1 or the Bcl3 gene inhibits skeletal muscle atrophy. J Clin Invest 114:1504–1511
Bartlett RJ, Secore SL, Singer JT et al (1996) Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector in to mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo. Cell Transplantation 5:411–419
Schertzer JD, Plant DR, Lynch GS (2006) Optimizing plasmid-based gene transfer for investigating skeletal muscle structure and function. Mol Ther 13:795–803
Wolff JA, Ludtke JJ, Acsadi G et al (1992) Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. HumMolGenet 1:363–369
Dickson G (1996) Gene transfer to muscle. BiochemSocTrans 24:514–519
Davis HL, Demeneix BA, Quantin B et al (1993) Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. HumGenTher 4:733–740
Mir LM, Bureau MF, Gehl J et al (1999) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96:4262–4267
Mathiesen I (1999) Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther 6:508–514
Aihara H, Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16:867–870
Dona M, Sandri M, Rossini K et al (2003) Functional in vivo gene transfer into the myofibers of adult skeletal muscle. Biochem Biophys Res Commun 312:1132–1138
Taylor J, Babbs CF, Alzghoul MB et al (2004) Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation. BMC Biotechnol 4:11
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Peters DG, Mitchell-Felton H, Kandarian SC (1999) Unloading induces transcriptional activation of the sarco(endo)plasmic reticulum Ca2+-ATPase 1 gene in muscle. Am J Physiol 276:C1218–1225
Zandi E, Rothwarf DM, Delhase M et al (1997) The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell 91:243–252
Reed SA, Senf SM, Cornwell EW et al (2011) Inhibition of IkappaB kinase alpha (IKKα) or IKKbeta (IKKβ) plus forkhead box O (Foxo) abolishes skeletal muscle atrophy. Biochem Biophys Res Commun 405:491–496
Durieux AC, Bonnefoy R, Busso T et al (2004) In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage. J Gene Med 6:809–816
Levy MY, Barron LG, Meyer KB et al (1996) Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood. GeneTher 3:201–211
Mitchell-Felton H, Kandarian SC (1999) Normalization of muscle plasmid uptake by Southern blot: application to SERCA1 promoter analysis. Am J Physiol 277:C1269–C1276
Wolff JA, Williams P, Acsadi G et al (1991) Conditions affecting direct gene transfer into rodent muscle in vivo. BioTechniques 11:474–485
Lee MJ, Cho SS, Jang HS et al (2002) Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation. Exp Mol Med 34:265–272
Davis HL, Whalen RG, Demeneix BA (1993) Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum Gene Ther 4:151–159
Gehl J, Sorensen TH, Nielsen K et al (1999) In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochim Biophys Acta 1428:233–240
Komamura K, Miyazaki J, Imai E et al (2008) Hepatocyte growth factor gene therapy for hypertension. In: Li S (ed) Methods in Molecular Biology, 1st edn. Human Press, New Jersey
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This work was supported by NIH grant AR41705.
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Wu, CL., Kandarian, S.C. (2012). Protein Overexpression in Skeletal Muscle Using Plasmid-Based Gene Transfer to Elucidate Mechanisms Controlling Fiber Size. In: DiMario, J. (eds) Myogenesis. Methods in Molecular Biology, vol 798. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-343-1_13
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DOI: https://doi.org/10.1007/978-1-61779-343-1_13
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