Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression
Tevz, Gregor, Pavlin, D., Kamensek, U., Kranjc, S., Mesojednik, S., Coer, A., Sersa, G., & Cemazar, M. (2008) Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression. Technology in Cancer Research and Treatment, 7(2), pp. 91-101.
Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.
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|Item Type:||Journal Article|
|Keywords:||And transfection efficiency, Electroporation, Electrotransfection, Green fluorescent protein, Inflammatory mononuclear cells infiltration, Long-term gene expression, Plasmid DNA, Skeletal muscle, adolescent, animal experiment, animal tissue, article, cell infiltration, controlled study, electric potential, female, gene delivery system, gene expression, gene targeting, gene transfer, genetic transfection, histology, mononuclear cell, mouse, muscle necrosis, nonhuman, protein synthesis, stereomicroscopy, transgene, Animals, Electric Stimulation, Green Fluorescent Proteins, Inflammation, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Muscle, Skeletal, Plasmids, Transfection|
|Divisions:||Current > Schools > School of Biomedical Sciences
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
|Deposited On:||08 Jul 2014 00:18|
|Last Modified:||08 Jul 2014 23:45|
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