Isolation and characterisation of the GnRH1 gene in Tilapia (Oreochromis niloticus) - with a view of producing transgenic sterile fish

Farahmand, Hamid (2003) Isolation and characterisation of the GnRH1 gene in Tilapia (Oreochromis niloticus) - with a view of producing transgenic sterile fish. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

In order to investigate the possibility of producing reversibly sterile tilapia (Oreochromis niloticus), the complete GnRH1 coding sequence with a substantial 5’ regulatory sequence has been isolated and characterized from a relevant genomic library for this species. Promoter efficacy has been tested using 0.6 kb of the GnRH promoter sequence driving a lacZ reporter gene in both primary cultures of spleen and transiently expressing zebrafish. In the cell transfection experiments, the average level of β-galactosidase activity was 2.1 fold higher (P<0.05) when compared to a control promoter-less vector in five independent culture and transfection experiments, indicating that the 0.6GnRH/lacZ construct is able to express in spleen cells. In addition, transient expression of the lacZ gene was detected in the putative brain region of 4 day old zebrafish embryos (Danio rerio) following egg microinjection with this construct. Isolation of the tilapia GnRH1 gene and its upstream sequences in this project has contributed to the further progress in producing sterility in tilapia via transgenic technology. This would entail the use of this promoter to drive GnRH1 antisense expression in order to ‘knock down’ the expression of the GnRH1 gene, resulting in sterility.

In addition, an alternative method of inducing sterility via tetraploid induction followed by triploidy (by crossing tetraploid fish with wild-type diploid fish) was investigated in this project. The induction of tetraploidy was carried out by means of thermal shock treatment on a transgenic line of tilapia containing a novel growth hormone gene and the lacZ gene. Production of tetraploid adult fish was not achieved although chromosome analyses and X-gal staining assays demonstrated the successful production of tetraploid transgenic tilapia as far as the hatching stage.

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