Research objectives and content
The zebrafish, Danio rerio, has become a popular model system for studying vertebrate development. Large-scale chemical mutagenesis screens have been performed in this system in which hundreds of developmentally important genes have been mutated. However, cloning of chemically mutated genes has to rely on the 'candidate gene' approach, or on positional cloning, which is time-consuming and laborious. Here, we propose to perform enhancer trap and gene trap screens in zebrafish. Cloning of genes identified by enhancer traps and gene traps is relatively easy as the affected genes are marked by the inserted DNA element. In addition, since the expression pattern of a reporter gene present in a gene trap construct reflects the expression pattern of the gene in which the construct has inserted, the search for the mutant phenotype will be more focused, allowing for identification of mild or easy to miss localized abnormalities. Previous work has shown that the Tc3 transposon of Caenorhabtitis elegans is functional in, zebrafish. Tc3 transposon mediated transgenesis will be optimized by testing several concentrations of transposase mRNA and by testing different transposon DNA constructs. To achieve high rates of germ-line transposition of the Tc3 transposon, fish lines that express the Tc3 transposase in the germ-line under control of the zebrafish vasa or the mouse protamine promoter will be constructed jump-starter lines). Lines transgenic for enhancer trap or gene trap constructs carrying the green fluorescent protein (GFP) reporter gene will be constructed, crossed to jump-starter lines and the progeny will be screened for expression of GFP. Lines showing an interesting region-specific pattern of expression in the developing nervous system will be further examined for neural patterning defects using marker gene expression analysis, morphological analysis and cell transplantation assays. For insertions showing interesting expression patterns or phenotypes an effort will be made towards molecular cloning of the relevant genes.
Training content (objective, benefit and expected impact)
My main scientific interest is in early vertebrate neural development, which I was studying in my previous lab using Xenopus as a model system. During my Ph.D. thesis I would like to ask questions in this field using a system offering genetic tools for analysis of developmental processes. I expect to be exposed to modern molecular genetics procedures and to significantly contribute to the establishment of enhancer trap and gene trap screens in zebrafish which will provide an important and efficient means to study vertebrate neural patterning. At the moment, the Institut Biologie in Freiburg is the only place in Europe where the approach of insertional mutagenesis in zebrafish is being pursued. Being part of this effort from its initial stages is an especially challenging experience for me.
Links with industry / industrial relevance (22)