of biological containment and gene flow in transgenic sterile fish
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.
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.
Husebye H., Collas P. and Aleström P., A functional study
of the salmon GnRH promoter.
Mol Mar Biol Biotech, 6,
1997, pp. 357-363.
Maclean N., Genetic manipulation of farmed fish, in
Biology of farmed fish, Sheffield Academic Press, Black K.
and Pickering A.D. (eds.), 1998, pp. 327-354.
Rahman M.A., Mak R., Ayad H., Smith A. and Maclean N., Expression
of a novel piscine growth hormone gene results in growth enhancement
in transgenic tilapia (Oreochromis niloticus).
Transgenic Research, 7, 1998, pp. 357-369.
Aleström P., Transgenic fish in food production, J.
Josefsson (ed.), Nordic Council of Ministers Bioethics Committee,
1998, pp. 54-60.
Martin S., Wallner W., Youngson A.F. and Smith T.J., Differential
expression of Atlantic salmon thyrotropin beta subunit mRNA and
its cDNA sequence.
Jnl. of Fish biology, 54,
1999, pp. 757-766.
December 1997 - November 2000
National University of Ireland
University of Southampton (UK)
Laboratoire de Biologie Moléculaire et de Génie Génétique
Laboratoire de Physiologie des Poissons
Laboratoire de Physiologie des Régulations
Norwegian College of Veterinary Medicine