Background and objectives ⁽¹⁾

The enormous potential benefits of transgenic fish technology in research and the aquaculture industry will not be achieved without effective isolation of genetically modified fish from the wild fish genetic pool. The possibility of transmission of transgenes to wild fish or of transgenic fish establishing themselves as permanent residents of an environmental ecosystem is the single most important negative consideration in applying this technology. This project provides a solution to this problem, without impairing the ability to transfer a gene of interest and a mechanism to test its effectiveness. The proposed strategy is also of possible relevance to other transgenic animals. Current approaches to genetic isolation involve either physical containment or induction of sterility by triploidisation. However, neither of these approaches is 100% effective, nor can the genetic changes induced by triploidy be accurately assessed, monitored or controlled. However, an effective means of inducing controlled reversible sterility is the complete and specific blockage of the reproductive system at the level of the brain.

The goal of this project is to develop molecular methods leading to the successful integration and expression of transgenes in fish leading to effective biological containment of these fish, while retaining the ability to transmit these traits to offspring under controlled conditions. This goal will be achieved by the production and analysis of stable lines of fish which have been rendered transgenically sterile by the inhibition of gonadotropin releasing hormone (GnRH) at the level of the brain using antisense mRNA technologies. We will also assess the effectiveness of the induced sterility by analysis of the reproductive endocrine system and the functionality of the gonads and by the evaluation of transgenic lines of fish, including regeneration of fertility in brood stock and analysis of stability of transgenes and sterility in offspring.

(1) This project was a direct follow-on of EC project BIO2-CT94-2039.


Approach and methodology

The primary objective of the project was the production and analysis of stable lines of fish, which had been rendered transgenically sterile by the inhibition of gonadotropin releasing hormone (GnRH) at the level of the brain. The strategy was to express GnRH antisense mRNAs ("anti-messages") under the control of a strong all-tissue promoter (Histone H3) to inhibit the biosynthesis of GnRH. The rationale was that the absence of GnRH peptides would result in a blockage of the hypothalamo-pituitary-gonad axis. Achievement of this objective required the development of molecular strategies to improve the efficiency of stable transgene integration and expression, in order to ensure effective sterility via transgenic induction. The second objective was to assess the effectiveness of the induced sterility in lines of transgenic fish (rainbow trout), by an analysis of the reproductive endocrine system and the functionality of the gonads. Furthermore, in order to produce stable lines of transgenic fish, methods to regenerate gametogenesis in these sterile transgenic fish (a key element required to produce fish lineages) were developed.

Main findings and outcome

The project successfully accomplished its major objective, which was the production of lines of transgenic fish (rainbow trout), which were rendered sterile through GnRH antisense mRNA. F1 and F2 progeny have been produced carrying GnRH antisense sequences under the control of the salmon histone H3 promoters and GnRH antisense mRNA expression was detected in a variety of tissues using an RT-PCR assay. The strongest expression was detected in immature gonads. F1 lines have been produced expressing the GnRH antisense, which contain sterile males (2.5 years old) that never produced sperm naturally. In these sterile male fish, induction of spermatogenesis was achieved using pituitary extract injections, which enabled the production of F1 and F2 generations of transgenic trout, containing GnRH antisense transgenes. These results show that inhibition of GnRH synthesis using antisense mRNA technology seems to be a safe and effective means of inducing sterility in genetically modified fish.

We have been successful in producing stable transgenic zebrafish by micro-injecting extremely low concentrations of expression vector DNA complexed to NLS peptides (~80% proportion of transgenic fish can be produced with as low as 100 copies injected).

Transgenic trout have been produced carrying GnRH antisense sequences under the control of the salmon GnRH and histone H3 promoters. Induction of spermatogenesis in F0 transgenic trout using pituitary extracts enabled the production of F1 and F2 generations of transgenic trout containing GnRH antisense transgenes. GnRH antisense mRNA expression has been detected in F2 fish, using an RT-PCR assay. Evaluation of antisense mRNA molecules and ribozymes for efficacy of inhibition of gene expression in a blue gill sunfish fibroblast cell line suggests that two of the ribozymes tested can decrease lacZ expression by 17-60% in co-transfected cells.

Conclusions

The successful accomplishment of the major goal of the project is extremely important in that it confirms that the strategy proposed for rendering fish sterile through inhibition of GnRH synthesis works as predicted. Thus the hypothesis and approach used were successful and stable lines of rainbow trout have been developed which are transgenically sterile but in which the sterility can be reversed under controlled conditions. The production of effectively sterilised fish, through genetic engineering also has broader implications in that once safe sterile transgenic lines have been produced, it will open up the opportunity of introducing other transgenes to improve the performance of fish, without the attendant risks of escape and interbreeding with wild fish populations. In addition, it confirms that antisense mRNA technology can be used to generate genetically modified fish and other farmed animals, with a range of economically valuable traits, through the inhibition of protein synthesis.