Abstract
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 P-galactosidase reporter genes was achieved either by direct injection or by particle bombardment with DNA-coated gold rnicroparticles. Luciferase expression was observed in homogenates of trout flank muscle and slun 2 d after injection of 10 to 100 pg of DNA per fish or bombardment of 1 pm gold particles coated with 5 to 25 pg DNA per fish at helium pressures ranging from 2750 to 12 400 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 P-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.
Highlights
In recent years, researchers studying fish diseases have increasingly used recombinant DNA technologies for the development of new vaccines for use in aquaculture (Leong 1993, Munn 1994, Press & Lillehaug 1995)
Among the applications of gene transfer, nucleic acid immunization is a promising new approach to vaccination (Tang et al 1992).Antigen-specific antibody production and protective immunity can be induced by delivering plasmid DNA, containing genes coding for antigenic proteins, into the tissues of live animals (Tang et al 1992, Cox et al 1993, Fynan et al 1993, Robinson et al 1993, Ulmer et al 1993,Wang et al 1993, Raz et al 1994, Sedegah et al 1994, Barry et al 1995, Manickan et al 1995)
In order to evaluate the efficiency of injection as a method for the delivery of DNA into live fish, w e performed injections of the plasmid pCMVtkluc into different tissues of the trout
Summary
Researchers studying fish diseases have increasingly used recombinant DNA technologies for the development of new vaccines for use in aquaculture (Leong 1993, Munn 1994, Press & Lillehaug 1995). Among the applications of gene transfer, nucleic acid immunization is a promising new approach to vaccination (Tang et al 1992).Antigen-specific antibody production and protective immunity can be induced by delivering plasmid DNA, containing genes coding for antigenic proteins, into the tissues of live animals (Tang et al 1992, Cox et al 1993, Fynan et al 1993, Robinson et al 1993, Ulmer et al 1993,Wang et al 1993, Raz et al 1994, Sedegah et al 1994, Barry et al 1995, Manickan et al 1995). O Inter-Research 1996 Resale of fulf article not permitted expressed by the animal cells and appears to stimulate persistent humoral and cellular responses without integration of plasmid into chromosomal DNA (Wolff et al 1992, Raz et al 1994). Particle bombardment and direct injection have been used to physically introduce genes into the cells and tissues of animals in vivo (Benvenisty & Reshef 1986, Wolff et al 1990, Hansen et al 1991, Williams et al 1991, Eisenbraun et al 1993, Sanford 1993).The direct delivery of gene constructs that encode for proteins from the influenza virus, the herpes sirnplex virus, and the malaria parasite has been shown to con-
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