M. Gomezchiarri et al., INTRODUCTION OF FOREIGN GENES INTO THE TISSUE OF LIVE FISH BY DIRECT-INJECTION AND PARTICLE BOMBARDMENT, Diseases of aquatic organisms, 27(1), 1996, pp. 5-12
We compared 2 methods of direct gene delivery into Live rainbow trout
Oncorhynchus mykiss tissue, with the final goal of developing DNA-base
d vaccines for bacterial diseases in salmonids. The introduction of pl
asmid constructs containing the luciferase and beta-galactosidase repo
rter genes was achieved either by direct injection or by particle bomb
ardment with DNA-coated gold microparticles. Luciferase expression was
observed in homogenates of trout flank muscle and skin 2 d after inje
ction of 10 to 100 mu g of DNA per fish or bombardment of 1 mu m gold
particles coated with 5 to 25 mu g DNA per fish at helium pressures ra
nging from 2750 to 12 400 kPa. Expression levels increased over 10 d a
nd persisted for at least 60 d after injection. For particle bombardme
nt, the most reproducible levels of luciferase expression were obtaine
d with the eye as a target (83% of fish positive versus 59% of fish po
sitive when the flank was the target). The levels of luciferase expres
sion observed after particle bombardment were significantly lower (t-t
est, p < 0.02) than the levels measured after direct injection. Immuno
histochemical analysis indicated beta-galactosidase gene expression in
muscle cells at the site of injection and in the dermis, epidermis an
d muscle after bombardment. These 2 methods may prove valuable for the
development of a new generation of DNA-based vaccines for fish.