Optimization of the Gal4/UAS transgenic tools in zebrafish
- 72 Downloads
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.
KeywordsGal4/UAS Toxicity Methylation Zebrafish
This research is funded by the Hunan Natural Science Foundation of China (2018JJ3371), the National Foundation of Natural Science of China (31572619), Science Foundation of Hunan university of Arts and Science (16BSQD47), and also by the development funds of Chinese central government to guide local science and technology (No.2017CT5013).
Compliance with ethical standards
This article does not involve human participants. All zebrafish experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals and were approved by Hunan University of Arts and Science.
Conflict of interest
The authors declare that they have no competing interests.
- Bougioukli S, Sugiyama O, Alluri R, Ortega B, Lieberman J (2017) Comparison of two lentiviral two-step transcriptional amplification systems using gal4ff or gal4vp16 transactivators for ex vivo regional gene therapy in bone repair. Mol Ther 25(5):214Google Scholar
- Kang T, Martins T, Sadowski I (1993) Wild type gal4 binds cooperatively to the gal1-10 UASG in vitro. J Biol Chem 268(13):9629–9635Google Scholar
- Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, lkeo K, Takeda H, Kawakami K (2008) Insertional mutagenesis by the tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like. Development 135(1):159–169CrossRefGoogle Scholar
- Ovchinnikov DA, van Zuylen WJ, Debats CE, Alexander KA, Kellie S, Hume DA (2008) Expression of gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using csf1r-gal4vp16/uas-ecfp double-transgenic mice. J Leukoc Biol 83(2):430–433CrossRefGoogle Scholar
- Zhang YS, Chen J, Cui XJ, Luo DJ, Xia H, Dai J, Zhu ZY, Hu W (2015) A controllable on-off strategy for the reproductive containment of fish. Sci Rep-UK 5(5):73–78Google Scholar