Functional EGFP-dystrophin fusion proteins for gene therapy vector development

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated minigene (6 kb) cDNAs are hampered by t he 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 c ostly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-lengt h human dystrophin were constructed. These constructs were tested by transf ection of Phoenix cells with Effectene, resulting after 48 h in a green flu orescent signal in 20% of cells, Analysis of the cell extracts by immunoblo tting with the use of a monoclonal antibody specific to the dystrophin C-te rminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-lengt h human dystrophin (450 kDa) fusion proteins. Moreover, following the in vi vo electroporation of the plasmids containing the EGFP-mini- and full-lengt h dystrophin in mouse muscles, both fluorescent proteins were observed in c ryostat sections in their normal location under the plasma membrane. This i ndicates that the fusion of EGFP to dystrophin or mini-dystrophin did not i nterfere with the normal localization of the protein. In conclusion, the fu sion of EGFP provides a good tool for the search of the best methods to int roduce mini- or full-length dystrophin cDNA in the cells tin vitro) or musc le fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.