Protein Engineering, Vol. 13, No. 9, 611-615,
September 2000
© 2000 Oxford University Press
Communication |
Functional EGFP–dystrophin fusion proteins for gene therapy vector development
Unité de Recherche en Génétique Humaine, Centre Hospitalier de l'Université Laval, CHUQ, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, G1V 4G2, Canada and 1 INSERM U 523, Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP–mini- (240 kDa) and EGFP–full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP–mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.
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