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Diseases of Aquatic Organisms

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DAO 27:5-12 (1996)  -  doi:10.3354/dao027005

Introduction of foreign genes into the tissue of live fish by direct injection and particle bombardment

Gómez-Chiarri M, Livingston SK, Muro-Cacho C, Sanders S, Levine RP

We compared 2 methods of direct gene delivery into live rainbow trout Oncorhynchus mykiss tissue, with the final goal of developing DNA-based vaccines for bacterial diseases in salmonids. The introduction of plasmid constructs containing the luciferase and beta-galactosidase reporter genes was achieved either by direct injection or by particle bombardment with DNA-coated gold microparticles. Luciferase expression was observed in homogenates of trout flank muscle and skin 2 d after injection of 10 to 100 µg of DNA per fish or bombardment of 1 µm gold particles coated with 5 to 25 µg DNA per fish at helium pressures ranging from 2750 to 12400 kPa. Expression levels increased over 10 d and persisted for at least 60 d after injection. For particle bombardment, the most reproducible levels of luciferase expression were obtained with the eye as a target (83% of fish positive versus 59% of fish positive when the flank was the target). The levels of luciferase expression observed after particle bombardment were significantly lower (t-test, p < 0.02) than the levels measured after direct injection. Immunohistochemical analysis indicated beta-galactosidase gene expression in muscle cells at the site of injection and in the dermis, epidermis and muscle after bombardment. These 2 methods may prove valuable for the development of a new generation of DNA-based vaccines for fish.

DNA-based immunization · Gene transfer · Particle bombardment · Injection · Rainbow trout · Reporter genes · Bacterial kidney disease · Furunculosis

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