Contents
-Home
-Welcome Letter
-Purpose of Workshop
-Program
-Presentations

Appendix
-Participants
-Steering Committee
-Program Committee

Workshop Report
-Preface
-Final Report

Introduction

Organisms into which foreign DNA (transgene) have been artificially introduced and stably integrated in host genomes are called "transgenic organisms" (for review: Gordon 1989 and Jaenish 1990). Since 1985, a wide range of transgenic fish species have been produced by microinjecting or electroporating desired foreign DNA into newly fertilized or unfertilized eggs (for review: Chen et al., 1990; Fletcher and Davies, 1991; Hackett 1993; Chen et al., 1995, 1996a, 1996b, 1998). Several important steps are routinely taken to produce a desired transgenic fish (Chen et al., 1998), and these steps are: (i) selecting an appropriate fish species; (ii) preparing a transgene construct; (iii) introducing the transgene construct into fish embryos; (iv) selecting and characterizing the resulting transgenic fish; and (v) breeding the desired transgenic fish into homozygotes. However, low rates of transgene integration and germ-line mosaicism in P₁ transgenic individuals produced by microinjection or electroporation are some of the severe limitations of these gene transfer methods (Chen et al., 1995, 1996a, 1996b, 1998). Furthermore, since the transgene is microinjected or electroporated into newly fertilized or unfertilized eggs, these gene transfer methods are only applicable to those animal species from which fertilized or unfertilized eggs can be readily obtained.

Burns and colleagues (Burns et al., 1993; Yee et al., 1994) have recently developed a series of new gene transfer vectors, broad host range (pantropic) replicative-defective retroviral vectors. These vectors contain a long terminal repeat (LTR) sequence of Moloney murine leukemia virus (MoMLV) and transgenes (e.g., neo^R or -gal) packaged in a viral envelop with the G-protein of vesicular stomatitis virus (VSV). These vectors were effective in infecting cell lines of fish, newt, Xenopus, and mosquito (Burns et al., 1993; Burns et al., 1994; Miyanohara et al., 1992; Matsubara et al., 1996), and newly fertilized finfish and shellfish eggs such as medaka, zebrafish and surf clam (Lin et al., 1994; Lu et al., 1996, 1997), since they contain the G-protein from the vesicular stomatitis virus (VSV) which binds to phospholipids in the cell membrane. Stable transgenic medaka and surf clams have been produced by electroporating these pantropic vectors into newly fertilized embryos (Lu et al., 1996, 1997). More recently, Sarmasik et al. (2001) have used these vectors to infect immature gonads of live-bearing fish and crayfish in situ and result in the production of transgenic individuals by crossing transformed animals with their untransformed counterparts.

A major drive of transgenic fish research came from biotechnological application to increase production of fish for human consumption (i.e., aquaculture). The worldwide supply of fish products depends upon harvesting of natural populations of finfish, shellfish and crustaceans. In 1992, however, the total annual worldwide harvest of fish products has surpassed the maximal potential level that nature can sustain, and many areas in the world have experienced shortages in fish populations. In order to cope with the worldwide demand of fish products, many countries have turned to aquaculture. Success in aquaculture depends on six factors: (a) complete control of the reproductive cycle of the fish species in culture; (b) excellent genetic background of the broodstocks; (c) efficient prevention and detection of disease infection; (d) thorough understanding of the optimal physiological, environmental and nutritional conditions for growth and development; (e) sufficient supply of excellent quality water; and (f) application of innovative management techniques. By improving these factors, the aquaculture industry has developed to a remarkable extent during the last two decades. To sustain this growth, however, newly developed technologies such as transgenesis will play an important role in revolutionizing aquaculture. The transgenic technology can be employed to enhance somatic growth, control reproductive cycles, improve feed compositions and feed conversion rates, produce new vaccines, and develop disease resistant and hardier brood stocks of finfish and shellfish for aquaculture.

Narrative

Transgene construct containing rainbow trout growth hormone or insect cecropin gene driven by a -actin promoter was electroporated into newly fertilized eggs of tilapia or medaka and presumptive transgenic fish were analyzed by PCR analysis. Transgenic founders were established by crossing P₁ transgenic animals with non-transgenic counterparts. Both F₁ and F₂ transgenic progeny were analyzed for transgene inheritance and transgene expression. Furthermore, the growth performance and reistance to disease infection were assessed in F₂ progeny.

Results and Conclusion

Cecropins are a family of antimicrobial peptides that were first discovered in silkmoth (Hyalophora cecropia), due to their potent bactericidal activity (Boman, 1994). Since then, these peptides have been identified in invertebrates and vertebrates. Cecropins are translated as precursors of 62-64 amino acid residues, and are processed intracellularly into mature peptides of 35-37 amino acid residues (Boman et al., 1991; Boman, 1995). Due to their unique structural features, cecropins can be readily incorporated into cellular membranes of bacteria, fungi and parasites which result in the formation of pores on the membrane and leading to the inevitable fate of death of pro- and eukaryotic pathogens via cellular lysis (Bechinger 1997).

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 pathogens (Merrifield et al., 1995; Kadono-Okuda et al., 1995; Vunnam et al., 1995) and protozoa (Rodriguez et al., 1995). 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).

Bacterial pathogens are the leading cause of fish disease outbreak in intensive aquaculture (Inglis and Hendrie, 1993; Thune et al., 1993). There are only a limited number of approved antibiotics for the treatment of diseased fish caused by bacterial pathogens (Post, 1987). Although recent advances in the development of recombinant subunit vaccines or DNA vaccines promise a potential bright future in controlling fish diseases, the difficulties of labor intensive and high stress in fish due to handling during the treatment process pose practical limitations of their usage (Fjalestad et al., 1993, Ganz, 1999). 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 as well. To confirm this hypothesis, we transferred cDNA of insect cecropin or pig cecropin-like peptide under the control of a cytomegalovirus (CMV) promoter 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 a zone inhibition assay. As shown in Fig. 1, a dose dependent bactericidal activity toward Aeromonas hydrophila and Pseudomonas fluorescens was observed in cecropins produced by the transfected CHSE-214 cells. These data suggest that the innate defense activity against bacterial pathogens might be enhanced if a cecropin gene were introduced into aquaculture important fish species. To test this feasibility, we have produced transgenic medaka (Oryzias latipes) carrying insect cecropin or pig cecropin-like gene driven by a CMV promoter by electroporation. To test the impact of cecropin transgene, F₂ cecropin transgenic fish were subjected to a challenge test at a LD50 dose with Pseudomonas fluorescens and Vibrio angularum, and the results are presented in Fig. 2. While about 50% of the non-transgenic medaka was 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 and its analogues to protect fish from infection by bacterial pathogens. Work is underway in our laboratory to produce disease resistant rainbow trout strains by transferring transgenes carrying cecropin peptides or synthetic cecropin analog peptides.

Infectious hematopoetic necrosis virus (IHNV) is a virulent viral pathogen that primarily infects the larvae or juveniles of salmon and rainbow trout. Outbreaks of this disease frequently wipe out the entire fish population in the impacted area. In a series of in vitro studies, we have observed that the replication of IHNV in CHSE-214 cells can be inhibited by synthetic cecropin and its analogues. These results point to the possibility that fish viral pathogens could be effectively controlled by these peptides.

Studies conducted in our laboratory showed that application of recombinant rainbow trout growth hormone to yearling rainbow trout by injection resulted in a dose-dependent growth enhancement. These results suggested that manipulating the endogenous levels of growth hormone might alter the somatic growth rate of rainbow trout or other fish species. To confirm this hypothesis, we have transferred rainbow trout growth hormone transgene into newly fertilized eggs of common carp, channel catfish and tilapia by electroporation. As shown in Fig. 3, significant growth enhancement was observed in resulting transgenic fish (for review: Chen et al., 1996b, 1998). These results point to the possibility of producing fast growing fish strain for aquaculture by the gene transfer technology.

Figure 1. Bactericidal activity of cecropin synthesized in CHSE-214 cells transfected with cecropin gene constructs. The bactericidal activity against P. fluorescens was determined zone inhibition assay. Medeia collected from transfected and non-transfected CHSE-214 cells were concentrated to: 1, 10X; 2, 20X, 3, 50X and 4, 100X. Each data point is the average of three independent determinations.

Figure 2: Cumulative mortality observed in transgenic and nontransgenic medaka following challenge with P.fluorescens (A) and V.anguillarum (B). In each challenge studies, 10 animals each of transgenic and non-transgneic animals are used and the dose of the bacterial pathogen used in studies will give 50% mortality in non-transgenic fish.

Figure 3. Growth hormone transgenic fish. Growth hormone transgene driven by the common carp -actin gene promoter was transferred into newly fertilized eggs. The transgenic fish grew substantially faster than their non-transgneic controls.

Recommendations

A critical question that we should address is the primary objective of developing aquaculture in U.S. Should we be developing the capability of mass production of fishery products by aquaculture to compete for the world market? Should we devote our resources to new and advanced aquaculture technologies for commercialization in the world market? Or should we concentrate our effort to develop niche market aquaculture products domestic and international markets. Due to constraint in land utilization and high labor cost, it is less feasible for U.S. to develop mass aquaculture production for competition in the world market. However, with the rich scientific resources in U.S., we are well suited for to developing advanced technologies for increasing aquaculture production that have high economic value in the world market. Furthermore, these advanced techniques enable us to capture the niche market of producing high quality and high economic value marine fish.

Traditionally the profit margin of aquaculture is very low. Consequently, any increase in the productivity of an aquaculture operation will certainly benefit the profit margin. Production of finfish, shellfish and crustaceans with enhanced somatic growth rate and/or increased resistance to infection by bacteria, fungi, parasites and viruses by the gene transfer technology promises to increase the productivity of aquaculture.

Short term

Effort should be placed in producing transgenic finfish and crustaceans carrying cecropin or synthetic cecropin analog transgene in order to breed disease resistant organisms, or carrying GH gene for enhanced somatic growth.

Mid-term

Studies conducted in cattle showed that mutation of myostatin gene will result the development of in double muscle (i.e., increase muscle growth). Although myostatin has also been identified in fish, it is not clear whether double muscle development will also occur in fish if the myostatin is mutated or the level of myostatin is greatly reduced. Therefore studies should be conducted to investigate this possibility. Transgenic fish with reduced levels of active myostatin gene product can be easily produced by introducing a dominant negative myostatin gene or an antisense gene. This approach should certainly be attempted. Another effort should be tried is the production of transgenic fish with -ketolase gene that will allow transgenic fish to produce high economic value carotenoids.

Long-term

Developing transgenic fish as bioreactors.

Acknowledgement

This research was supported by grants from NSF (IBN-9723529 and IBN-0078067, USDA (#CONS-9803641 and CONTR # 58-1930-0-009) and Connecticut Sea Grant College (R/A 18) to TTC.

References

Ganz, T. (1999) Defensins and Host defense. Science. 286: 420-421.