of transgenic technology in fish: assessment and reduction of risks
The objective of this project is to assess and reduce the risks that could
emerge from fish genetically altered by recombinant DNA technology. Our
approach is to improve the technologies that are available to produce
transgenic fish and to minimise methodological problems, including the
production of unnecessary genetically modified organisms. This will be
done by developing strategies to control integration and expression of
the introduced genes and by improving the methods for analysing transgenic
fish. As an alternative to stable, irreversible alteration of the germline,
somatic gene transfer as a transient alteration will be rigorously evaluated
and technologically improved. It is not the aim of the project to produce
transgenic fish with altered characteristics, to genetically manipulate
commercial species or to release genetically modified organisms into the
Approach and methodology
A method based on the polymerase chain reaction was developed for efficient
testing of suspected transgenic fish in large numbers that is sensitive
enough to detect even subgenomic amounts of foreign DNA. The method shows
also high specificity, allowing not only the detection of foreign DNA
material from other organisms routinely used in recombinant DNA technology
(e.g. bacterial sequences), but also if the transgene is of fish origin.
A second method has been developed on the basis of a very sensitive reporter
gene, the green fluorescent protein, for sorting putative transgenics
at a very early stage of the procedure.
To obtain fidelity of transgene expression is still a significant problem.
Work has been started to isolate so-called locus control regions (LCR)
and matrix attachment sites (MAR) from fish genes. Both such types of
sequences are known from other organisms to be instrumental in guaranteeing
quantitatively and qualitatively correct expression of endogenous genes
and in preventing transgenes from being silenced.
To improve existing ES cell culture systems derived from the medaka, a
small freshwater fish, the inhibition of differentiation through a specific
selection scheme was used. The medaka is one of the most widely used model
Main findings and outcome
Functionally analogous or homologous LCR sequences in the fish genome
have been shown to exist in this project by generating a model fish line,
where a defective pigmentation gene could be corrected leading to the
rescue of the normal wildtype phenotype. For switching on and off of fish
genes through exogeneous agents, the tetracylin-regulated gene system
was for the first time introduced into fish cells in vitro.
Rather than up-regulating the level of a certain gene, which is still
today the most frequent motivation for producing a transgenic fish, there
is an increasing number of reasons why the expression of an endogenous
gene should be down-regulated. We tested the use of antimessages and ribosomes
to regulate protein expression. No indication has so far been obtained
that these two methods are appropriate for use in fish.
Methods were established to introduce genes into a large marine fish and
a small freshwater fish species. In addition, in the laboratory fish Xiphophorus,
another tissue, namely melanoma, was shown to be also a suitable target
for somatic gene transfer. The method was refined so that it is now not
only qualitative but also allows precise quantification of promoter activity.
The system can now be used for tissue-specific gene expression. Crucial
differences compared to mammals were noted in the mechanisms and biological
phenomena underlying gene expression after somatic gene transfer. This
excludes the simple transfer to fish of methods developed for instance
in mammals and underlines the necessity for further activity in this research
We have seen evidence that fish and mammals might share similar control
mechanisms for maintaining the undifferentiated stage of embryonic stem
cells, which is remarkable because of the many differences noted so far
between mouse and fish ES cells. The differentiation inhibition approach
can also now be used to re-evaluate other species, which so far were refractory
to the technique for establishing embryonic stem cell cultures. Methods
for introducing DNA into ES cells in culture have been optimised and a
test gene for a first homologous recombination experiment was analysed.
We have refined and extended techniques designed to generate transgenic
fish lines in a controllable and reproducible fashion. Our work has developed
methods for manipulating the germline of fish and has contributed to the
understanding of molecular events controlling gene expression and regulation
in the germline. These findings may also be relevant for the genetic manipulation
of other organisms.
Fu L.J., Mambrini M., Perrot E., Chourrout D., Stable and
full rescue of the pigmentation in a medaka albino mutant by transfer
of a 17 kb genomic clone containing the medaka tyrosinase gene.
2000, pp. 205-211.
Hong Y., Winkler C., Schartl M., Production of medakafish
chimeras from a stable embryonic stem cell line.
Proc Natl. Aca Sci, 95,
1998, pp. 3679-3684.
Joly J.S., Kress C., Vanderputte M., Bourrat F., Chourrout D.,
Irradiation of fish embryos prior to blastomere transfer
boosts the colonisation of their gonads by donor-derived garnetes.
Mol Reprod Dev, 53, 1999, pp. 394-397.
Schartl M., Gómez A., Hong Y., Gene transfer in fish:
approaches, progress and perspectives, Third European Marine
Science and Technology Conference, 1999, pp. 171-188.
January 1999 December 2001
University of Wuerzburg (DE)
Laboratoire de Génétique des Poissons
R. Di Lauro
Stazione Zoologica A. Dohrn
University of Southampton (UK)
Instituto de Acuicultura de Torre de la Sal
SARS International Centre for Molecular Marine Biology HiB