The first and major objective is to produce fish which are transgenically sterile by inhibition of gonadotropin releasing hormone (GnRH) synthesis using antisense GnRH mRNA. The second objective is to develop methods to optimise nuclear localisation, integration and expression of transgenes in fish embryos and to produce fish which are transgenic for GnRH antisense message as well as chosen reporter genes. Tissue-specific and all-tissue expressing promoters will be compared for expression of GnRH antisense and reporter genes. The third objective is 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 sterility.
During the course of the project, substantial progress was made in all four task areas of the project and a number of significant scientific breakthroughs were achieved. The major achievements within each task were as follows:
1. Isolation and characterisation of fish genes and promoters
Novel salmon and tilapia genes and promoters (Histone H3, ribosomal protein L18, hsp70), were isolated, characterised and shown to be expressed at high levels in a wide variety of tissues, confirming their all-tissue expression. The tilapia and zebrafish homologues of the salmon GnRH gene were isolated, characterised and compared to the salmon GnRH gene. Constructs expressing lac Z and CAT reporter genes and GnRH sense and antisense genes (constructed from the salmon GnRH gene sequence), under the control of strong all-tissue promoters (H3, carp beta-actin) and the salmon GnRH promoter were tested and shown to function correctly in vitro and in vivo. The strong all-tissue promoter constructs functioned as expected while low lac Z expression levels were obtained specifically in GnRH cells from the GnRH promoter. The carp beta-actin promoter was shown to be significantly more efficient in fish (tilapia and zebrafish) than its rat homologue, while the species specificity was found to reside in intron 1. An enhancer co-injection strategy has been developed for testing and identifying strong positive regulatory elements in a transient expression assay in zebrafish. This method enables the identification of novel enhancer sequences and combinations of positive regulatory elements as well as the determination of their tissue-specificity in vivo.
2. Optimisation of stable transgene integration and expression
Significant improvements in the efficiency of integration and expression of transgenes in fish were obtained using nuclear localisation signal (NLS) sequences and co-injection methods. A method utilising NLS sequences was developed and found to improve the efficiency of nuclear import of transgenes (100 fold increase). NLS also facilitates the expression and transmission of transgenes. Improvements in integration and gene expression have also been achieved by co-injection of enhancer sequences (carp beta-actin and myosin heavy chain enhancers).
3. Transgenic fish production and analysis
CAT and lac Z reporter genes were shown to be expressed in the predicted manner in trout, tilapia and zebrafish. GnRH (sense and antisense) genes, under the control of Histone H3, carp beta-actin and GnRH promoters were microinjected into trout, tilapia and zebrafish. Salmon GnRH transgene expression (transient) under the control of the salmon GnRH and H3 promoters was confirmed in trout embryos using a GnRH-specific ELISA. Tissue-specific expression was obtained using the GnRH promoter while expression was detected in a wide range of tissues using the H3 promoter. GnRH antisense expressing were injected into fish embryos and transgenic F0 progeny obtained. The F0 zebrafish have reached maturity and have been used to produce F1 generation fish which will be examined for GnRH expression, gonad development and sterility. GnRH antisense mRNA was detected by RT-PCR and reduced GnRH levels were found in the pituitaries of GnRH antisense-containing F0 trout, compared to non-transgenic fish.
4. Analysis of GnRH system in trout and tilapia
When inducing transgenic sterility in fish, it is essential to gain a basic understanding of which function of the brain pituitary gonadal axis could be altered to induce sterility. GnRH expression in developing embryos detected by Immunocytochemistry using antiserum directed against GnRH, indicates that GnRH expressing cells are detectable 52-57 days post-fertilisation in trout and after a few days in tilapia. In tilapia as in tout, the GnRH neurons migrate rapidly through the olfactory nerves to the olfactory bulbs and preoptic area. These studies which also identified two major forms of GnRH in tilapia and their distribution within the brain, enable comparison with transgenic sterile fish when they are produced.
MAJOR SCIENTIFIC BREAKTHROUGHS:
1. Isolation and characterisation of novel salmonid, tilapia and zebrafish all-tissue and tissue-specific promoters (Histone H3, L18, hsp70, GnRH) and confirmation of their correct function in vitro and in vivo. These will be of general use in transgenic fish research.
2. Development of a novel co-injection method to identify regulatory elements and their tissue specificity. This approach can be used to identify enhancers and silencers with particular tissue-specificity, enabling controlled transhgene expression in all cell types. This approach will be of use in all transgenic animal types, including mammals.
3. Significant improvements in methods to maximise integration and expression of transgenes in vivo, by the use of NLS sequences and co-injection of enhancers. These developments will be of major and general value in transgenic fish research and in other transgenic animal research.
4. Detection of GnRH transgene expression in vivo under the control of tissue-specific and all-tissue promoters. This will enable the sensitive monitoring of changes in GnRH levels as a result of GnRH antagonism by antisense mRNA.
5. Demonstration that 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
6. Detection of GnRH expressing cells, molecular forms, distribution and characterisation of the GnRH system during development in trout and tilapia. This data is essential in analysing the development and function of the GnRH system in transgenic sterile fish.
Funding SchemeCSC - Cost-sharing contracts
SO9 3TU Southampton