Optimizing Plasmid-Based Gene Transfer for Investigating Skeletal Muscle Structure and Function

Department of Physiology, The University of Melbourne, Melbourne, VIC 3010, Australia.
Molecular Therapy (Impact Factor: 6.43). 05/2006; 13(4):795-803. DOI: 10.1016/j.ymthe.2005.09.019
Source: PubMed

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.

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Available from: Jonathan D Schertzer, Apr 16, 2014
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    • "Each TA muscle was injected with 30 lL of vector (25 lg of vector, 5 lg of GFP), and 5 mm electrodes (BTX Gentrodes, Harvard Apparatus , Holliston, MA, USA) were applied to either side of injection site. Three 20-ms pulses of 100 mV were applied using an ECM-830 electroporator (Harvard Apparatus) according to a protocol previously reported to cause only moderate damage to the tissue (Schertzer et al. 2006); polarity of electrodes was reversed and electroporation was repeated. Skin was closed over muscle using wound clips, and 1 lL of buprenorphine was injected subcutaneously for pain relief. "
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    ABSTRACT: Six1 is necessary for the genesis of several tissues, but in adults it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow-to-fast fiber type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown. The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector. Muscles were collected at 2 or 14 days after transfection for Six1 and myosin heavy chain (MHC) expression analysis. C2 C12 mouse myoblasts were grown in standard conditions. Cells were co-transfected with MHC promoter vectors and Six1 expression vectors. Cells were harvested after 4 days of differentiation. In vivo, the Six1 siRNA caused a decreased expression of Six1,1.8-fold (±0.1, p<0.05). With decreased Six1, MHC IIB expression decreased 2.7-fold (±0.7, p=0.04). Proportion of muscle fibers expressing MHC IIB decreased (45.3±4.8% versus 65.1±7.3% in control group, p=0.04), and total area expressing MHC IIB decreased with decreased Six1 (59.6±4.3% versus 75.2±5.4% in control group, p<0.05). Decreased Six1 increased MHC IIA expression 1.9-fold (±0.3, p=0.04). In vitro, Six1 overexpression increased promoter activation of MHC IIB 2.9-fold (±0.3, p<0.01). Six1 knockdown repressed MHC IIB promoter 2.9-fold (±0.1, p<0.05) and MHC IIX 3.7-fold (±0.08, p<0.01). Six1 knockdown caused a fast to slow shift in MHC isoform, and this was confirmed by promoter activity of MHC genes. Six1 may ultimately control the contractile and metabolic properties that define muscle fiber phenotype. This article is protected by copyright. All rights reserved.
    Acta Physiologica 09/2013; 210(2). DOI:10.1111/apha.12168 · 4.25 Impact Factor
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    • "Co-immunostaining for laminin g-1 ensured that plasmid expression was confined to skeletal muscle fibers and not within extracellular space. Gene transfection efficiency was determined by calculating the cross sectional area of GFP positive muscle fibers expressed as a percentage of the total cross sectional area (Schertzer et al, 2006). All constructs revealed a transfection efficiency of 58.6–78.5% (Supporting Information Fig S6D). "
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    EMBO Molecular Medicine 01/2013; 5(1). DOI:10.1002/emmm.201201443 · 8.25 Impact Factor
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    • "Indeed, ectopic Akt expression is sufficient for increasing the size of myofibers in adult mice (Bodine et al., 2001; Takahashi et al., 2002; Lai et al., 2004). Although the effect of IGF-1 on skeletal muscle mass mainly depends on development, IGF-1 can lead to increase skeletal muscle mass even in adulthood (Adams and McCue, 1998; Barton-Davis et al., 1998; Alzghoul et al., 2004; Schertzer et al., 2006). "
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    ABSTRACT: Skeletal muscle comprises approximately 40% of body weight, and is important for locomotion, as well as for metabolic homeostasis. Adult skeletal muscle mass is maintained by a fine balance between muscle protein synthesis and degradation. In response to cytokines, nutrients, and mechanical stimuli, skeletal muscle mass is increased (hypertrophy), whereas skeletal muscle mass is decreased (atrophy) in a variety of conditions, including cancer cachexia, starvation, immobilization, aging, and neuromuscular disorders. Recent studies have determined two important signaling pathways involved in skeletal muscle mass. The insulin-like growth factor-1 (IGF-1)/Akt pathway increases skeletal muscle mass via stimulation of protein synthesis and inhibition of protein degradation. By contrast, myostatin signaling negatively regulates skeletal muscle mass by reducing protein synthesis. In addition, the discovery of microRNAs as novel regulators of gene expression has provided new insights into a multitude of biological processes, especially in skeletal muscle physiology. We summarize here the current knowledge of microRNAs in the regulation of skeletal muscle hypertrophy, focusing on the IGF-1/Akt pathway and myostatin signaling.
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