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Mol Ther. 2004 Oct;10(4):679-87.

Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts.

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Unité de Recherche en Génétique Humaine, Centre de Recherche du CHUL, CHUQ, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada, G1V 4G2.


Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

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