Fertilized eggs of loach (Misgurnus fossilis), rainbow trout (Salmo gairdneri) and zebrafish (Brachydanio rerio) were bombarded with high‐velocity tungsten microprojectiles covered with plasmid DNA containing sequences of β‐galactosidase and neomycin phosphotransferase genes. About 70% of the eggs survived the bombardment. The activity of both transferred genes was revealed in the fish developed from the bombarded eggs. Neomycin phosphotransferase gene sequences were detected by means of PCR amplification and Southern hybridization in the total DNA of zebrafish that survived after G418 treatment.
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.
Mouse cells of developing embryos at the 24 cell, morula and blastocyst stages, were bombarded by high velocity tungsten microprojectiles. About 70% of developing embryos survived the bombardment. The general embryo structure did not change as a result of the bombardment. Penetration of the tungsten microparticles into the embryo cell nuclei was found at all stages being investigated, and tungsten particle localization on mitotic chromosomes was demonstrated. The total DNA of the mice born from the bombarded embryos was analyzed by dot-blot hybridization and PCR with post-hybridization. The most important results were obtained in experiments with blastocysts. In three cases of blastocyst bombardment, the presence of transferred plasmid DNA (pSV3-neo) was revealed. Transfected cells were shown to be located in the fetal membrane as well as in the embryo. The bombardment of mouse culture cells resulted in their transfection and the production of G418-resistant clones.
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