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|Title:||Transgene expression in mononuclear muscle cells not infiltrating inflammatory cells following intramuscular plasmid gene electrotransfer|
Christopher G. Huggins
James A.M. Shaw
University of Newcastle upon Tyne, Faculty of Medicine
Newcastle University, United Kingdom
|Keywords:||Biochemistry, Genetics and Molecular Biology;Medicine;Pharmacology, Toxicology and Pharmaceutics|
|Citation:||Journal of Gene Medicine. Vol.12, No.4 (2010), 377-384|
|Abstract:||Background: In situ electroporation-assisted intramuscular plasmid DNA delivery offers high efficiency for therapeutic protein replacement. Expression may be impaired by an immune response against the plasmid or transgenic protein. Expression of the transgene in non-muscle cells may increase the immune response. Gene transfer efficiency and phenotypic identification of intramuscular transgene-expressing mononuclear cells was studied following electroporation-mediated plasmid delivery. Methods: Plasmids expressing β-galactosidase (pVR1012-βgal) or enhanced green fluorescent protein (eGFP) (pVR1012-eGFP) were electrotransferred into rat tibialis anterior muscles. Both transfection efficiency and the inflammatory response were determined in pVR1012-βgal-injected muscles by β-galactosidase and haematoxylin and eosin staining of muscles 7 days post-plasmid injection. Muscles injected with pVR1012-eGFP were stained for CD3, CD68 and desmin at 24 and 48 h post-injection to determine whether mononuclear cells expressing eGFP were of immune or myogenic origin. Results: With electroporation, β-galactosidase expression was significantly enhanced by up to ten-fold compared to plasmid injection without electroporation. A large area of regenerating muscle fibres and inflammatory cell infiltration was found in electroporated plasmid-injected muscle. No eGFP expression was found in CD3- or CD68-positive cells. Small mononuclear cells expressing eGFP showed negative staining for CD3 and CD68, but all stained positive for desmin. Conclusions: In situ electroporation enhanced transfection efficiency of plasmid DNA delivery into muscle. Alongside its advantage for improving gene transfer, electroporation led to an increased inflammatory response and muscle damage. Mononuclear cells in muscle were transfected with plasmid and expressed the transgene. These cells were of myogenic origin with no evidence of transgene expression in infiltrating inflammatory cells. Copyright © 2010 John Wiley & Sons, Ltd.|
|Appears in Collections:||Scopus 2006-2010|
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