Applied Microbiology and Biotechnology

, Volume 103, Issue 4, pp 1789–1799 | Cite as

Optimization of the Gal4/UAS transgenic tools in zebrafish

  • Yunsheng ZhangEmail author
  • Jiawei Ouyang
  • Jingrong Qie
  • Gongyuan Zhang
  • Liangguo Liu
  • Pinhong Yang
Applied genetics and molecular biotechnology


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.


Gal4/UAS Toxicity Methylation Zebrafish 


Funding information

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.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yunsheng Zhang
    • 1
    Email author
  • Jiawei Ouyang
    • 1
  • Jingrong Qie
    • 1
  • Gongyuan Zhang
    • 1
  • Liangguo Liu
    • 1
  • Pinhong Yang
    • 1
  1. 1.Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher EducationHunan University of Arts and ScienceChangdePeople’s Republic of China

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