containment of transgenic fish and risk assessment of inter-species gene
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
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
N. & Rahman A., Transgenic Fish, in Animals with
Novel Genes, Cambridge Univ. Press, N. Maclean (ed.), 1995,
Poncelet A.C., Yaron Z., Levavi-Sivan B., Martial J.A. and Muller
M., Regulation of prolactin gene expression in fishes,
in Recent advances in marine biotechnology, Nagabhushanan
R., Thompson M.-F. and Fingerman M. (eds.), Oxford and IBH Publishing
Co., New Delhi, 1996, pp. 383-405.
Husebye H., Collas P. and Aleström P., A functional study
of the salmon GnRH promoter.
Mol. Mar. Biol. Biotech., 6, 1997, pp. 357-363.
Bailhache T., Uzbekova S., Breton B., Jego P., Localisation
of gonadotropin-releasing hormone neurons in the brain of the tilapia
(Oreochromis niloticus) during development and in mature
Gen. Comp. Endocr., 1997.
Hanley S., Muller F., Maclean N., Uzbekova S., Prunet P., Breton
B. and Smith T.J., Isolation and functional analysis of the
histone H3 promoter from Atlantic salmon (Salmo salar L.).
Mol. Mar. Biol. Biotechnol., 7 (3), 1998, pp. 165-172.
November 1994 - April 1997
National University of Ireland
of the project
This project was continued in EC project BIO4-CT970554.
University of Southampton (UK)
University of Liège (BE)
Laboratoire de Physiologie des Poissons
Laboratoire de Physiologie des Régulations
Norwegian College of Veterinary Medicine