Development of serine recombinases as tools for genome engineering in zebrafish

Site specific recombinases are proteins that promote reciprocal and conservative recombination between specific DNA sequences. In both fruit flies and mice site specific recombinases have been used to turn off genes in particular tissues and to manipulate chromosome structure. This type of manipulation is known as genome engineering. Genome engineering in fruit flies and mice has relied on reversible site specific recombinases. Over the past few years however we have shown that a new type of site specific recombinase; one that promotes a unidrectional or irreversible reaction, is very active in vertebrate cells in cell culture and can be used for elaborate genome engineering reactions in these cells. We would now like to use these enzymes for genome engineering in zebrafish . We wish to establish a methodology for the efficient construction of animals in which particular tissues are labelled with fluorescent proteins and to establish a system for gene targeting by homologous recombination.

Zebrafish has rapidly become a model organism of central importance in biomedical research. Zebrafish have been used in several large scale mutagenesis programs that have resulted in the isolation of thousands of mutations, many in genes involved in developmental processes that are important in human biology. We propose to use genome engineering to develop two sets of genome engineering tools that will extend the utility of zebrafish as a model organism. 1/ Embryological studies often require the use of organisms containing specifically labelled tissues. We aim to develop a promoter swap strategy that will allow the routine construction of zebrafish with specifically labelled tissues. 2/ Gene targeting is a technique that is fundamental to the analysis of gene function. In complex multi-cellular eukaryotes genes that play important roles in development typically do so in a variety of different tissues and at several different time points in the development of the organism. Tissue specific or conditionally active mutations are therefore particularly important for the analysis of gene function. The techniques that are currently used for the study of gene function in zebrafish such as morpholinos or TILLING cannot be used for tissue specific analysis. However gene targeting may be used to introduce conditionally active and tissue specific mutations into the genome. Thus model organisms, such as yeast and mouse, in which gene targeting is practical are particularly important in experimental biology. Gene targeting has not been developed in zebrafish.We propose to use unidirectional site specific recombinases as tools to establish a methodology for gene targeting in zebrafish that is essentially the same as that developed for gene targeting in Drosophila by Golic and colleagues.