Despite the many advantages of zebrafish as a genetic system, there is currently no technology to manipulate its genome through targeted mutagenesis.
We aim to overcome this barrier in zebrafish, through manipulation of the activity of RecQ helicases in the germline. There is evidence in human, mouse, and chicken that the enzymes encoded by the Bloom syndrome (BLM) and RECQL5 genes normally function to regulate homologous recombination during DNA damage repair. Reduction of their normal activities leads to an increase in spontaneous sister chromatid exchange and homologue recombination, and also correlates with increased frequency of extrachromosomal homologous recombination. We propose to transiently repress BLM and RECQL5 functions in the zebrafish embryo to allow manipulations of the genome, including targeted mutagenesis in the germline. We have identified the zebrafish orthologues of BLM and RECQL5 and verified their expression in the early embryo. First, we will measure the ability of putative dominant negative (dn) mutations of blm and recqIS to repress endogenous gene function. We have shown that injection of dnblm RNA into embryos heterozygous for the pigment mutation golden induces recombination between homologues, detectable as clones of mutant cells in the retinal pigment epithelium at 3 days of development. We will use this assay to determine the best combination of injected RNAs to transiently repress helicase function while still allowing normal development. We then propose to target dnRNAs to primordial germ cells through the addition of the nanosl 3'UTR, confining their activity to the germline of injected embryos. Fish in which helicase function in the germline has been transiently suppressed will be raised and screened for germ cell survival, reduced fertility, and induced chromosomal rearrangements or other mutations. This will establish the feasibility of recovering targeted mutations in the next generation. Finally, we have made targeting constructs to introduce mutations in 2 loci. The first will insert GFP into the dead end locus, which encodes a putative RNA helicase expressed specifically in germ cells; positive targeting events can be scored as fluorescent germ cells in injected larvae. The second will introduce a dominant point mutation into the somitobun gene; embryos derived from oocytes carrying the mutation will display a characteristic patterning defect. These assays will be used to optimize the protocol for gene targeting. Once established, these techniques for repressing function of RecQ helicases could facilitate many manipulations of the zebrafish genome, such as creation of genetic mosaics; generation of germ cells homozygous for maternal effect mutations, and ultimately germline homologous recombination. ? ? ?
