The objective of the project is to study the molecular mechanisms of trout defences against pathogens. The goals are (i) the generation of molecular markers predicting the capacity of fish immune response in relation to disease resistance (ii) the generation of novel cell lines of the immune system for in vitro functional studies (iii) the validation of these markers by gene regulation studies in vivo and in vitro (iv) an interference with pathogen infection using genes encoding these markers, other genes involved in fish defences, and genes of the pathogens themselves. The project also intends to create an industrial platform on fish genetics and diseases.
State of progress
Homology cloning in the rainbow trout has resulted in full length cDNAs which encode the interleukin 1 beta, a chemokine receptor related to the receptor of interleukin-8, the cyclooxygenase 2 (COX2) and the proteasome subunit LMP2 (up regulated in mammals by interferons). One genomic clone containing the interleukin-1 beta gene has also been obtained by PCR and characterised. Functional studies have already revealed potential roles for two of these factors in fish defences since (i) three IL1beta transcripts are induced by infection with a bacterial pathogen and stimulation by PHA (ii) COX 2 is expressed in several lymphoid tissues and the cDNA contains motifs known to interfere with cytokine functions. Differential cloning from mitogen-stimulated and non stimulated leukocytes has been attempted by several methods. Suppressive subtractive hybridisation looks more promising and has generated numerous candidate clones. Finally, the expression of the platyfish Xmrk receptor in a stimulatable form in mouse lymphocyte cell lines has revealed interactions with eight known genes of the cytokine-related intracellular signalling and two known cytokine-induced genes. One of these genes (JAK1) has been cloned in trout during this project.
Attempts to immortalise trout leucocytes by transfection with several known platyfish oncogenes have now been initiated, after earlier experiments have demonstrated that the platyfish Xmrk oncogene interacts with the signal transduction machinery of mouse lymphocytes. Peptides have been designed from the known sequences of the trout T-cell receptor and used to generate monoclonal antibodies potentially able to mark trout T-cells. So far, none of them recognises a substantial fraction of peripherical blood cells or of thymocytes. As an alternative and original approach, experiments have shown that regulatory elements of a mammalian T-cell specific gene (CD2) drive the expression of a reporter gene in the thymocytes of transgenic trouts. Stable lines of such fish are now generated, and transgenic trouts expressing an exogenous transmembrane protein on their T-cells are tentatively produced. Candidate regulatory sequences from the trout TCR genes are also cloned in parallel. Finally, an important methodological investment has been engaged to increase the feasibility of enhancer trap screens to identify new cell populations of the medaka immune system and results in (i) new possibilities to detect transgenics even at low frequencies by transfer of the tyrosinase gene (ii) considerable improvement of the cell reimplantation techniques for future in vitro enhancer traps (iii) two minimal promoters which will be incorporated into enhancer trap constructs.
Functional studies of several genes cloned in the project already permit to foresee novel cytokine-based strategies to positively manipulate the trout immune system by either genetic or non-genetic routes. Several "natural" or mutant versions of sequences encoding variable regions of MAb reacting with the VHSV G protein have been cloned and used to engineer single chain antibodies (scAbs). Some of these molecules were shown to bind and even neutralise several VHSV strains, and are currently used for passive immunisation of normal fish and for transgenic experiments. Transfection of fish cell lines with vectors including two VHSV genes in sense orientation has resulted in expression of the encoded proteins. These vectors have been successfully used for DNA vaccination and they are currently tested in transgenic experiments.
The four industrial subcontractors have prepared the basis of a platform in the field of fish genetics and diseases, and a workshop associating scientists and the fish farming industry.
IV Major Achievements
- Cloning/identification of several trout cytokine, cytokine-related and cytokine-induced genes
- Stable transgenic trouts expressing a reporter gene in thymocytes; Methodological bases for an enhancer trap screen in medaka.
- Evidences for the implication in the immune response of several genes cloned in the project; Engineering of several reactive single-chain antibodies against the VHSV G protein.
- Expression of several VHSV gene constructs in fish cells; Similar constructs already efficient for DNA vaccination; Transgenic trouts available with an anti-sense construct derived from the VHSV M1 gene.
- Basis for an industrial platform in the field of fish genetics and diseases.
Description of work
Task 1 consists of the homology cloning of cDNAs and genes encoding cytokine genes. Due to the relatively low conservation of cytokines, these genes will also be searched by differential cloning between stimulated and non-stimulated leucocytes.
Task 2 consists of the recognition and isolation of novel cell lines of the fish immune system, which are so far available essentially in the channel catfish. To this end, four approaches are implemented, three of which relying upon gene transfer in vitro and in vivo.
Task 3 consists of the implementation of various functional studies of genes isolated in the project, to determine their role in the immune system and also to see whether a modification of their expression pattern can stimulate the fish defences. A major part of this task is to determine whether Pathogen-Derived Protection can work in transgenic fish as it does in transgenic plants.
Task 4 consists in preparing the creation of a platform of industrials:
Funding SchemeCSC - Cost-sharing contracts
8200 N Aarhus C
AB9 2TN Aberdeen
751 23 Uppsala