Background and objectives

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. We explored the feasibility of inducing controlled reversible sterility through the complete and specific blockage of the reproductive system at the level of the brain.

The ultimate goal of this project was to develop molecular methods leading to the effective biological containment of farmed fish, with the ability to reverse the sterility under controlled conditions. The major objectives of the project were to produce sterile fish by inhibition of gonadotropin releasing hormone (GnRH) synthesis using antisense GnRH mRNA and to develop methods to optimise expression of transgenes in fish. In doing this, we also hope to gain an understanding of the physiological and endocrinological function of the brain pituitary gonadal axis in fish and how the GnRH system can be altered to induce reversible sterility.


Approach and methodology

The primary objective of the project was the production and analysis of stable lines of fish which were rendered transgenically sterile by the inhibition of gonadotropin releasing hormone (GnRH) at the level of the brain. This objective is achieved by the expression of GnRH antisense mRNAs to inhibit the biosynthesis of GnRH. Absence of GnRH results in a blockage of the hypothalamo-pituitary-gonad axis. Realisation of this objective requires development of molecular strategies to improve the efficiency of stable transgene integration and expression, in order to ensure effective sterility via transgenic induction. We then assessed the effectiveness of the induced sterility by analysis of the reproductive endocrine system and the functionality of the gonads. 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

We isolated and characterised novel salmonid, tilapia and zebrafish all-tissue and tissue-specific promoters (Histone H3, L18, hsp70, GnRH) and confirmed their correct function in vitro and in vivo. These are of general use in transgenic fish research. We also developed a novel co-injection method to identify regulatory elements and their tissue specificity. This approach is used to identify enhancers and silencers with particular tissue-specificity, enabling controlled transgene expression in all cell types. This is of vital importance in the construction of safe genetically modified fish and other organisms.

We could detect GnRH transgene expression in vivo under the control of tissue-specific and all-tissue promoters, which enables the sensitive monitoring of changes in GnRH levels as a result of GnRH antagonism by antisense mRNA. GnRH antisense expression in transgenic F0 trout resulted in a decrease in GnRH levels in pituitary cells. This suggests that the antisense GnRH approach will be successful, resulting in sterile F1 generation transgenic fish.

Conclusions

We obtained data which strongly supports the transgenic antisense expression system as a safe and effective means of inducing sterility in genetically modified fish, a necessary stage in the control of released genetically modified organisms. The positive results and outcome from this project led to a subsequent successful proposal to the EU Biotechnology programme (BIO4-CT97-0554).