title: Bacterial beta-galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection.
abstract: Ballistic transfection, based on cell and tissue bombardment by the tungsten and gold microparticles covered with the gene DNA, was used for the delivery of a bacterial beta-galactosidase and a full-length cDNA copy of the human dystrophin genes into mouse skeletal muscles. CMV-lacZ, SV40-lacZ, LTR-lacZneo and full-length cDNA dystrophin (pDMD-1, approximately 16 kb) in eukaryotic expression vector pJ OMEGA driven by mouse leukaemia virus promotor (pMLVDy) were used throughout the studies. Musculus glutaeus superficialis of C57BL/6J and quadriceps femoris of mdx male mice were opened surgically under anesthesia and bombarded by means of the gene-gun technique originally developed by us. Different mixtures of gold and tungsten particles at ratios of 4:1, 1:1, 1:4 were applied. X-gal assay revealed marked beta-gal activity, both in total muscles and whole muscle fibers on histological sections, up to three months after transfection. The most intensive staining was observed after SV40-lacZ delivery. No staining was detected with LTR-lacZneo DNA as well as in untreated muscles. The higher tungsten particle concentration in the bombardment mixture correlated with more intense X-gal staining. At the gold/tungsten ratio of 1:4 the microparticles penetrated the musculus glutaeus superficialis and transfected the underlying musculus glutaeus medius as well. Immuno-cytochemical assay for human dystrophin revealed dystrophin positive myofibers (DPM) in the bombarded area up to two months after transfection. The proportion of DMP varied from 2.5% on day 17 up two 5% on day 60 after bombardment compared to only 0.5% in the control mdx mice. These results suggest the applicability of particle bombardment for gene delivery into muscle fibers.
authors.affiliation: Engelhardt Institute of Molecular Biology, Moscow, Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Zelenin
authors.forename: V A
authors.initials: VA
authors.lastname: Kolesnikov
authors.forename: O A
authors.initials: OA
authors.lastname: Tarasenko
authors.forename: R A
authors.initials: RA
authors.lastname: Shafei
authors.forename: I A
authors.initials: IA
authors.lastname: Zelenina
authors.forename: V V
authors.initials: VV
authors.lastname: Mikhailov
authors.forename: M L
authors.initials: ML
authors.lastname: Semenova
authors.forename: D V
authors.initials: DV
authors.lastname: Kovalenko
authors.forename: O V
authors.initials: OV
authors.lastname: Artemyeva
authors.forename: T E
authors.initials: TE
authors.lastname: Ivaschenko
authors.forename: O V
authors.initials: OV
authors.lastname: Evgrafov
authors.forename: G
authors.initials: G
authors.lastname: Dickson
authors.forename: V S
authors.initials: VS
authors.lastname: Baranovand
mesh_terms: D000818:Animals; D019470:Biolistics / Q000379:methods; D018076:DNA, Complementary; D016189:Dystrophin / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D009052:Leukemia Virus, Murine; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D016189:Dystrophin; D001616:beta-Galactosidase