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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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Transgenic fish stay
in the pond


T. Smith,
National Diagnostics Centre,
National University of Ireland (IE)
B. Breton and P. Prunet,
Laboratoire de Physiologie des Poissons,
INRA-Rennes (FR)

Through recent revolutionary advances in biotechnology, transgenic fish now offer the opportunity for major advances in scientific knowledge as a model species, and significant economic improvements to the aquaculture industry. The potential to produce transgenic lines of fish with improved performance characteristics has already started to become a reality. Transgenic fish, including Atlantic salmon and Tilapia, with improved growth rates have been produced, and comparative growth trials with non-transgenic fish have shown the benefits of transgenic technology to both researchers and producers. Other potential applications of transgenic fish concern the production of disease-resistant fish or fish with an improved carbohydrate metabolism.

However, the application of gene transfer technology to commercial fish species raises major concerns among consumers and environmentalists about the benefit of their use in aquaculture and the possible risks associated with genetically modified fish. One major question is the possibility of transgenic fish interbreeding with wild native stocks, leading to undesirable ecological impacts and dilution of the wild fish genetic pool. Escape of farmed fish into the sea is relatively common and can induce gene diffusion to wild fish. The risk of interbreeding concerns genetically selected strains of fish as much as genetically engineered transgenic fish, but public concern is much greater when genetically engineered fish are potentially involved. The possibility of the transfer of transgenes to wild fish or of transgenic fish establishing themselves as permanent residents of an environmental ecosystem is the most important negative consideration in applying this technology to fish culture. Therefore, the enormous commercial potential benefits of transgenic fish technology in research and in aquaculture will not be achieved without effective isolation of genetically modified fish from the wild fish genetic pool.

For further development of the use of transgenic fish in the aquaculture industry, there are two options: total physical containment of genetically modified fish or development of new methodologies for biological containment of transgenic fish.

Total physical containment of farmed fish is an unrealistic option for economic reasons given the huge cost of enclosed systems, particularly for sea-based facilities. Biological containment involves the production of sterile lines of fish to avoid possible gene transfer from escaped farm fish. This has been achieved by polyploidisation of the genome and although this strategy is currently used in aquaculture fish, it is not 100% effective in inducing sterility. Even a very small risk of failure in the induction of sterility, leading to some fertile animals, is unacceptable when dealing with genetically modified fish. The required goal is absolute 100% sterility. Therefore, an effective means of inducing controlled reversible sterility is the complete and specific blockage of the reproductive system at the level of the brain.

An innovative approach to blocking the fish reproductive system, which was the basis of two EC research projects (BIO2-CT94-2039 and BIO4-CT97-0554), involved the inhibition of sexual maturation using genetic engineering in a way that could be reversed under controlled conditions to produce brood stocks. These projects were based on the hypothesis that inhibition of gonadotropin-releasing hormone (GnRH), the neuropeptide responsible for the control of gonadotropin synthesis and secretion from the brain, and essential for sexual maturation, would lead to sterility. The rationale for this approach was that induced sterility could be reversed under controlled conditions through treatment with GnRH, thus enabling the production of fertile brood stock, essential to maintain and pass on commercially beneficial traits to offspring. The strategy adopted involved the expression of antisense messenger RNA (mRNA) to GnRH under the control of a strong promoter (Histone H3) in the same cells as those where GnRH is expressed, to inhibit the translation of GnRH mRNA to protein.

The outcome of this interdisciplinary research programme involving fish physiologists, endocrinologists, molecular biologists and geneticists was the successful generation of transgenically sterile fish (rainbow trout), which can be returned to fertility under controlled conditions by injection of GnRH to fish at the time of sexual maturation. The strategy of expressing antisense mRNA to GnRH has been shown to work and render fish sterile, yet the sterility can be reversed through treatment with GnRH, thereby proving the hypothesis correct. Thus, the inhibition of GnRH mRNA through antisense technology seems to be a safe and effective means of inducing sterility in genetically modified fish. The success of this project and of the antisense approach is extremely important, not only for the induction of sterility in fish, but also in proving that antisense mRNA technology can be applied successfully to inhibit the function of other endogenous fish genes which may lead to the introduction of other valuable traits in farmed fish. The outcome also has important implications beyond transgenic fish, as it shows that similar approaches can be used in other farm animal species to produce valuable traits.

This research also demonstrated that the effect of a transgene, integrated into the genome, depends upon the number of integrated copies and its site of insertion. In other words, as for all introduced transgenes, the efficiency of the technique is limited by the current state of the art in the field. The requirement for improvements to transgenic technologies and the efficiency of the process of transgenesis is being met by another EC project (FAIR-CT98-3482), which has as its major objectives the assessment and reduction of the risks associated with transgenic fish through transgenic technology improvements. This project is focused on the development of molecular strategies to control the integration and expression of introduced genes and methods for analysing transgenic fish. Successfully controlling the process and sites of transgene integration and expression will enable the targeted integration of transgenes, including GnRH antisense mRNA, resulting in the production of transgenic fish stocks with well-defined functional characteristics and stable properties. The success of this project will also have huge positive implications for the broad application of this important technology, not only in fish but in all farmed animal species.

These projects on biosafety of transgenic fish and others on wider aspects of animal transgenesis are at the cutting edge of research where breakthroughs need to be followed up and translated into economic advantage. In particular, we need a better understanding of transmission, expression and stability of the transgenes. In the transgenically sterile trout, further molecular and physiological studies are required to determine the stability of the transgenic lines and to determine if the approach is reproducible and predictable. It is also important to improve the efficiency of the process of transgene integration and expression. For this, studies on model species such as medaka and zebrafish will be invaluable because of their short generation times and ease of manipulation with genetic engineering technologies. In all cases, stable lines of transgenic fish are essential for a complete understanding of the biological impact of integrated transgenes on the whole organism.

Potentially adverse consequences of transgenes include the possibility that several phenotypic traits might be modified by the same transgene. This is especially true in the case of growth hormone, used to produce faster-growing fish but which not only stimulates growth and age of sexual maturity but also adaptation to sea water (smoltification). Thus, in terms of future transgenesis research, it is of importance to characterise the new phenotype of transgenic animals in detail to identify any unpredicted and possibly adverse pleiotropic effects due to the transgene. Such studies will be also essential for biosafety reasons. Among the various questions raised by transgenesis, one possible objection is that transgene integration could lead to the production of toxic molecules, evoking health concerns for consumers. An acceptable answer for this problem would be to carry out both critical trials for possible toxic effects in human and non-human, but also to have a complete physiological characterisation of the phenotype of transgenic fish, particularly those to be produced for commercial use.

In considering transgenic research and its applications, the possibility of access by genetically modified animals to the environment and their interaction with the environment must also be taken into consideration. There is actually no field study to evaluate the global impact of new introduced species or transgenes in a given environment. Multidisciplinary field studies in reconstituted environment structure such as mesocosms might also be developed. However, because of the complex interactions present in such experimental tools it could be recommended to develop a step-by-step procedure using more and more complex systems from aquaria to mesocosms, each new transgene being a special case. In planning for any such research, the utilisation of genetically modified animals which are stably and effectively sterile is essential.

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