Trapping of maternal genes in zebrafish

Tomoya Kotani and Koichi Kawakami

National Institute of Genetics, Mishima, Japan

　We have been developing a gene trap method in zebrafish using the Tol2 transposon system. The insertions of a transposon-based gene trap vector containing a splice acceptor and the promoter-less GFP gene caused the GFP-expression in temporally and spatially restricted patterns during development. In the pilot experiment, we identified 36 fish lines with unique GFP patterns and found that 10 out of them expressed GFP also in the fertilized eggs. Genes expressed maternally are thought to play essential roles in early development. We hypothesize our gene trap system should be useful to identify novel genes expressed maternally and involved in early development. In order to determine whether maternal genes are indeed trapped in these fish and to identify novel maternal genes, we analyzed the fish lines expressing GFP in the fertilized eggs.
　The gene trap fish line, SAG20, expressed GFP in the fertilized eggs and ubiquitously during gastrulation. Then the GFP expression was restricted in notochord at 30 hpf. The trapped sequence was identical to ENSDART00000014161, a predicted gene by the genome sequencing. The SAG20 insertion was found to trap the first non-coding exon. The product of the novel gene has a coiled coil domain in the N-terminal region but no homology with any functional protein.
　The SAGp53B expressed GFP in the fertilized eggs and ubiquitously throughout development, but particularly stronger in the CNS region during 24 to 54 hpf. 5’RACE revealed that the SAGp53B insertion trapped an exon of an unknown gene.
　The SAGm18B expressed GFP in the fertilized eggs and ubiquitously during gastrulation. Then the GFP expression was restricted in the CNS region at 30 hpf. The SAGm18B insertion was found to locate within an internal intron of a gene for succinyl CoA: 3-oxoacid CoA-transferase.
　The SAGp49A expressed GFP in the fertilized eggs and ubiquitously during gastrulation. Then the GFP expression was restricted on the surface of the head region after 30 hpf. The SAGp49A insertion was mapped on chromosome 24.
　The SAG14 expressed GFP in the fertilized eggs. Then the GFP expression was restricted in the midbrain and hindbrain boundary at 30 hpf. The locus of SAG14 insertion was elucidated.
　Characterization of these trapped genes, other fish lines with maternal-GFP expression, and the homozygous female phenotypes are currently under way.