INTRODUCTION OF FOREIGN GENES INTO THE TISSUE OF LIVE FISH BY DIRECT-INJECTION AND PARTICLE BOMBARDMENT

Citation
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
Citations number
27
Categorie Soggetti
Veterinary Sciences","Marine & Freshwater Biology
ISSN journal
01775103
Volume
27
Issue
1
Year of publication
1996
Pages
5 - 12
Database
ISI
SICI code
0177-5103(1996)27:1<5:IOFGIT>2.0.ZU;2-G
Abstract
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.