Background and objectives

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 environment.


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 systems.
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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 field.

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.
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Conclusions

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.