Abstract
Summary: Fish into which foreign DNA (transgene) have been artificially introduced and integrated in their genomes are called transgenic fish. Since the mid 1980s, introduction of desired foreign DNA into unfertilized or newly fertilized eggs by microinjection or electroporation has produced many species of transgenic fish. More recently, infecting newly fertilized eggs or the immature gonads of male fish with replication-defective pantropic retroviral vectors carrying the desired foreign DNA has also produced transgenic finfish. These transgenic fish could serve as excellent experimental models for basic scientific investigations as well as biotechnological applications. In this paper, we will review the current status of the transgenic fish technology and provide data from our own research to show the feasibility of producing disease resistant fish strains for aquaculture via manipulation of anti microbial peptide genes.
INTRODUCTION
The worldwide supply of fishery products traditionally depends on the natural population of finfish, shellfish, crustaceans and macroalgae of fresh water and marine sources. Due to the consequence of rapid increase in the world-wide consumption of fishery products, uncontrolled fishing activity, and poor management and restocking efforts, the levels of the total annual harvest of fish products has already surpassed the maximal annual sustainable level of 90 million metric tons in 1992.
INTRODUCTION
In addition, rapid accumulation of chemical pollutants in aquatic environments, as the results of increasing industrial activities, has caused further decline of the worldwide fishery productivity. The Food and Agriculture Organization (FAO) of the United Nations reported that while the worldwide demand for fishery products increased to 120 million metric tons by the of year 2000, the wild catch decreased to the level of about 85 million metric tons. As a result, aquaculture (fish farming) increased from the 1994 level of 13.9 million metric tons to approximately 35 million metric tons in six years, in order to make up the difference.
INTRODUCTION
Projection by FAO showed that aquaculture production have to further increase to 52 million metric tons by 2010 and 77 million metric tons by 20251. consequently, the possibility of conducting gene transfer studies in these fish species by the conventional microinjection or electroporation method is entirely eliminated. In many aquaculture important crustacean species, the transfer of spermatotheca from males to females initiates fertilization, and by the time embryos are released from females, they are in advanced developmental stages.
INTRODUCTION
Similarly, transfer of foreign genes into these crustacean species cannot be achieved by the conventional microinjection and electroporation methods. Therefore, alternative methods suitable for producing transgenic live-bearing fish and crustaceans would be highly desirable. Table 1 shows patterns of proviral sequence transmission to the F, progeny in the desert guppy and the crayfish by crossing P, transgenics to their counterparts of untreated animals. The F, progeny derived from the treated adults (P, animals) carrying neon transgene, ranging from several to 70%.
INTRODUCTION
These results suggest that, in both species of animals, only a fraction of the germline in each P, animal was transformed by the proviral vectors. When the F, animals derived from different P1 transgenic lines were mated with non-transgenic controls, about 50% of the resulting F2 progeny were shown to carry the neon transgene ( These applications have been extensively reviewed". Another drive of transgenic fish research came from biotechnological application to increase production of fish for human consumption (i.e., aquaculture). Prevention of disease outbreak is one of the prerequisites for the success of aquaculture. Bacterial pathogens are the leading cause of fish disease outbreak in intensive aquaculture23,24. There are only a limited number of approved antibiotics for the treatment of diseased fish caused by bacterial pathogens25. Although recent advances in the development of recombinant subunit vaccines or DNA vaccines promise a potential bright future in controlling fish disease, the difficulties of labor intensive and high stress in fish due to handling during the treatment process pose practical limitations of their usage26,27.
INTRODUCTION
Cecropins are a family of antimicrobial peptides that were first discovered in silkmoth (Hyalophora cecropia), due to their potent bactericidal activity". Since then, these peptides have been identified in invertebrates and vertebrates.
INTRODUCTION
Cecropins are translated as precursors of 62-64 amino acid residues, and are processed intracellularly into mature peptides of 35-37 amino acid residues 29,30. Due to their unique structural features, cecropins can be readily incorporated into cellular membranes of bacteria, fungi and parasites which results in the formation of pores on the membrane and leading to the inevitable fate of death of pro and eukaryotic pathogens via cellular lysis31.
INTRODUCTION
Several cercropin analogs have also been designed and synthesized. These analogs are as effective, or even more potent, than the native compounds against animal and plant bacterial pathogens32-34 and protozoa35. Genes of cecropins and their analogs have been used in the production of transgenic whole plants (e.g., potato and tobacco) with increased resistance to infection by bacterial or fungal pathogens (Hassan et al. and 1993; Jia et al., 1993) . Since cecropins and other antimicrobial peptides are effective in controlling bacterial pathogens in insects and plants, they might be effective in controlling bacterial pathogens in fish. To confirm this hypothesis, we transferred cDNA of insect cecropin or pig cecropin-like peptide under the control of a cytomegalovirus (CMV) promoter ( Fig.  1) into a chinook salmon embryonic cell line (CHSE-214). Cecropin peptide produced by the transfected CHSE-214 cells were tested for its bactericidal activity toward known fish bacterial pathogens such as Aeromonas hydrophila, Pseudomonas fluorescens and Vibrio angularum in the inhibition zone assay. To test this feasibility, we have produced transgenic medaka (Oryzias latipes) carrying insect cecropin or pig cecropin-like transgene driven by a CMV promoter (Fig. 1) by electroporation. To test the impact of cecropin transgene in the resulting transgenic fish, F2 cecropin transgenic fish were subjected to a challenge test at a LD50 dose with Pseudomonas fluorescens and Vibrio anguilarum, and the representative results are presented in Fig. 3 . While about 50% of the non-transgenic medaka were killed by the bacterial pathogens, less than 10% of the transgenic animals were killed by the bacterial pathogens. These results clearly demonstrate the efficacy of using cecropin or its analogues to protect fish from infection by bacterial pathogens. Fig.3 : Cumulative mortality observed in transgenic and non-transgenic medaka following challenge with P.fluorescens and V.anguillarum. In each challenge study, 10 animals each of transgenic and non transgneic animals are used and the dose of the bacterial pathogen used in the study will give 50% mortality in non-transgenic fish.
INTRODUCTION
Viruses are another important pathogens causing fish diseases. Using infectious hematopoetic necrosis virus (IHNV), a virulent viral pathogen that primarily infects the larvae or juveniles of salmon and rainbow trout, as a model we have investigated the effect of cecropin or its analog on controlling the propagation of viral particles in CHSE-214 cells. In a series of in vitro studies (Fig.  4) , we have observed that the replication of IHNV in CHSE-214 cells was inhibited by cecropin and its analogues. These results point to the possibility that fish viral pathogen could also be effectively controlled by these peptides. 

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