Development of theGal4-enhancer trap system using the Tol2 transposable element in zebrafish

Kazuhide Asakawa1 , Aki Ito1 , Akihiro Urasaki1 , Tomoya Kotani1 , Saori Nagayoshi1 , Yasuyuki Kishimoto1 , Masahiko Hibi2 , Koichi Kawakami1

1. National Institute of Genetics,

 To understand the mechanism of organogenesis in vertebrate, we developed the Gal4-UAS system in zebrafish using the Tol2 transposon system.
 First, by using the Tol2 transposable element, we constructed an enhancer trap vector hsp-gal4, which contains the yeast transcription factor GAL4 gene placed under the heat shock promoter, and reporter vectors, UAS-EGFP and UAS-DsRED. Then, we established transgenic zebrafish lines carrying these transposon vectors by Tol2-mediated transgenesis. We found that, upon heat shock, embryos obtained from crosses between the hsp-gal4 fish and these reporter lines could express EGFP and DsRed ubiquitously, suggesting that Gal4 expressed from hsp-gal4 can induce expression of a gene placed under UAS.
 Next, to determine whether Gal4-enhancer trapping is feasible, we created random insertions of the hsp-gal4 vector in the genome. In a pilot screen, we identified 30 lines that showed unique Gal4 expression patterns during embryonic stages by crossing the hsp-gal4 fish with the reporter lines. Inverse PCR and the following database search allowed us to map rapidly the insertion sites of hsp-gal4 on the genome and to identify candidate genes regulated by the trapped enhancer. These Gal4 lines should be used to express a gene of interest in the specific organs and tissues.