Development of Gal4-enhancer trap system using the Tol2 transposon in zebrafish

Kazuhide Asakawa1 , Aki Ito1 , Akihiro Urasaki1 , Ghislaine Morvan1 , Tomoya Kotani1 , Takeshi Sasaki2 , Saori Nagayoshi1 , Yasuyuki Kishimoto1 , Masahiko Hibi3 , Koichi Kawakami1

i1Nat. Inst. Genetics, 2Nat. Inst. Basic Biology, 3RIKEN CDBj

@Ectopic gene expression mediated by the Gal4-UAS method has been a quite effective system to elucidate gene function in organogenesis in Drosophila. The availability of such gene expression systems in vertebrate, however, is limited since highly efficient method for transgenesis has not been developed until recently.
@To understand the mechanism of organogenesis in vertebrate, we developed the Gal4-UAS system in zebrafish using Tol2 transposon. First, to identify organ-specific enhancer elements, we constructed an enhancer trap vector using the Tol2 transposable element that contains the gene encoding a yeast transcription factor Gal4 fused to GFP under the heat shock promoter. We found that embryos obtained from the cross between hsp-gfp-gal4 transgenic fish and the transgenic fish carrying UAS-DsRed displayed the expression of DsRed upon heat shock, suggesting that GFP-Gal4 can direct the expression of gene placed under UAS. Second, we performed a pilot screen for hsp-gfp-gal4 fish that showed a heat shock-independent expression of GFP-Gal4 during early embryonic stages. The screen identified 15 fish lines that showed unique GFP-Gal4 expression patterns at normal temperatures. We confirmed that UAS-DsRed was specifically induced in a tissue where GFP-Gal4 was expressed, indicating that our Gal4-UAS system in zebrafish should be useful to analyze the function of genes in vertebrate organogenesis.