High-efficiency gene electrotransfer into skeletal muscle: Description and physiological applicability of a new pulse generator

Laboratoire de Physiologie, Groupe Physiologie et Physiopathologie de l'Exercice et du Handicap, Groupement d'Intérêt Public-Exercice Sport Santé, Faculté de Médecine J. Lisfranc, 15 rue Ambroise Paré, Saint-Etienne Cedex, France.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 09/2002; 296(2):443-50. DOI: 10.1016/S0006-291X(02)00901-4
Source: PubMed


Efficiency and reproducibility of gene electrotransfer depend on the electrical specifications provided by the pulse generator, such as pulse duration, pulse number, pulse frequency, pulse combination, and current intensity. Here, we describe the performances of GET42, a pulse generator specifically designed for gene electrotransfer into skeletal muscle. Expression of beta-galactosidase in the Tibialis anterior muscle of Sprague-Dawley male rats was increased 250-fold by GET42 compared to DNA injection alone. Combination of high and low current intensity pulses further increased transfection efficiency (400-fold compared to DNA injection without electrotransfer). Varying degrees of muscle necrosis were observed after gene electrotransfer. Nevertheless, muscle necrosis was dramatically reduced after optimization of cumulated pulse duration without significant reduction in transfection efficiency. Physiological applicability was illustrated by the analysis of cytochrome c promoter transactivation. In conclusion, GET42 has proven to be a reliable and efficient pulse generator for gene electrotransfer experiments, and provides a powerful mean to study in vivo the regulation of gene expression.

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Available from: Anne-Cécile Durieux, Jun 11, 2014
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    • "Seven days after gene electrotransfer, the animals were anesthetized and the tibialis anterior muscles were removed. Protein isolation and measurement of firefly luciferase activity were performed as described previously [45]. To correct for interindividual variations in transfection efficiency, luciferase activity was normalized to β-galactosidase activity [45]. "
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    ABSTRACT: Sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase, has emerged as a main determinant of whole body homeostasis in mammals by regulating a large spectrum of transcriptional regulators in metabolically relevant tissue such as liver, adipose tissue and skeletal muscle. Sterol regulatory element binding protein (SREBP)-1c is a transcription factor that controls the expression of genes related to fatty acid and triglyceride synthesis in tissues with high lipid synthesis rates such as adipose tissue and liver. Previous studies indicate that SIRT1 can regulate the expression and function of SREBP-1c in liver. In the present study, we determined whether SIRT1 regulates SREBP-1c expression in skeletal muscle. SREBP-1c mRNA and protein levels were decreased in the gastrocnemius muscle of mice harboring deletion of the catalytic domain of SIRT1 (SIRT1(Δex4/Δex4) mice). By contrast, adenoviral expression of SIRT1 in human myotubes increased SREBP-1c mRNA and protein levels. Importantly, SREBP-1c promoter transactivation, which was significantly increased in response to SIRT1 overexpression by gene electrotransfer in skeletal muscle, was completely abolished when liver X receptor (LXR) response elements were deleted. Finally, our in vivo data from SIRT1(Δex4/Δex4) mice and in vitro data from human myotubes overexpressing SIRT1 show that SIRT1 regulates LXR acetylation in skeletal muscle cells. This suggests a possible mechanism by which the regulation of SREBP-1c gene expression by SIRT1 may require the deacetylation of LXR transcription factors.
    PLoS ONE 09/2012; 7(9):e43490. DOI:10.1371/journal.pone.0043490 · 3.23 Impact Factor
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    • "Indeed, an efficient transfection into skeletal muscles and tumors required the application of limited number of long pulses (several ms) at a low frequency (1 Hz). But, a very high number of repetitive short but stronger pulses (several μs) at a high frequency (kHz) give a high level of gene expression into the same tissues (Lucas et al. 2002; Vicat et al. 2000; Rizzuto et al. 1999; Mir et al. 1999; Durieux et al. 2002). A suitable protocol using a short high voltage pulse (kV/cm, μs) followed by several longer low voltage pulses (V/cm, ms) at a low frequency (1 Hz) has been proposed to aid gene delivery into skeletal muscle (Bureau et al. 2000). "
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    ABSTRACT: Gene electrotransfer can be obtained not just on single cells in diluted suspension. For more than 10 years, this is a quasi routine strategy in tissue on the living animal and a few clinical trials have now been approved. New problems have been brought by the close contacts of cells in tissue both on the local field distribution and on the access of DNA to target cells. They need to be solved to provide a further improvement in the efficacy and safety of protein expression. There is a competition between gene transfer and cell destruction. Nevertheless, present results are indicative that electrotransfer is a promising approach for gene therapy. High level and long-lived expression of proteins can be obtained in muscles. This is used for a successful method of electrovaccination.
    Biophysical Reviews 11/2009; 1(4-DOI 10.1007/s12551-009-0019-2):185-191. DOI:10.1007/s12551-009-0019-2
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    • "Transfection efficiency was calculated as the percentage of the area stained in blue over the whole muscle cross-sectional area [7] [30]. Muscle damage was assessed from Hemalun –Eosin – Safran (HES) staining and expressed as the percentage of damaged area over the whole crosssectional area [7] [30]. 2.6. "
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    ABSTRACT: We determined over a 3-week period some of the factors that may influence the kinetic of gene expression following in vivo gene electrotransfer. Histochemical analysis of beta-galactosidase and biochemical analysis of luciferase expressions were used to determine reporter gene activity in the Tibialis anterior muscles of young Sprague-Dawley male rats. Transfection efficiency peaked 5 days after gene electrotransfer and then exponentially decreased to reach non-detectable levels at day 28. Reduction of muscle damage by decreasing the amount of DNA injected or the cumulated pulse duration did not improve the kinetic of gene expression. Electrotransfer of luciferase expression plasmids driven either by viral or mammalian promoters rather show that most of the decrease in transgene expression was related to promoter origin/strength. By regulating the amount of transgene expression, the promoter origin/strength could modulate the immune response triggered against the foreign protein and ultimately the kinetic of transgene expression.
    Biochimica et Biophysica Acta 11/2005; 1725(3):403-9. DOI:10.1016/j.bbagen.2005.06.016 · 4.66 Impact Factor
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