Optimization of the Gal4/UAS transgenic tools in zebrafish.

The Gal4/UAS system provides a powerful tool to analyze the function of genes. The system has been employed extensively in zebrafish; however, cytotoxicity of Gal4 and methylation of UAS can hinder future applications of Gal4/UAS in zebrafish. In this study, we provide quantitative data on the cytotoxicity of Gal4-FF and KalTA4 in zebrafish embryos. A better balance between induction efficiency and toxicity was shown when the injection dosage was 20 pg for Gal4-FF and 30 pg for KalTA4. We tested the DNA methylation of UAS in different copies (3×, 5×, 7×, 9×, 11×, and 14×), and the results showed, for the first time, that the degree of UAS methylation increases with the increase in the copy number of UAS. We detected insertions of the Tol2-mediated transgene in the Gal4 line and found as many as three sites of insertion, on average; only about 20% of individuals contained single-site insertion in F1 generation. We suggested that the screening of Gal4 lines with single-site insertion is essential when Tol2-mediated Gal4 transgenic lines are created. Moreover, we designed a novel 5 × non-repetitive UAS (5 × nrUAS) to reduce the appeal of multicopy UAS as a target for methylation. Excitingly, the 5 × nrUAS is less prone to methylation compared to 5 × UAS. We hope the results will facilitate the future application of the Gal4/UAS system in zebrafish research.