PUBLICATION
Optimization of the Gal4/UAS transgenic tools in zebrafish
- Authors
- Zhang, Y., Ouyang, J., Qie, J., Zhang, G., Liu, L., Yang, P.
- ID
- ZDB-PUB-190108-5
- Date
- 2019
- Source
- Applied Microbiology and Biotechnology 103(4): 1789-1799 (Journal)
- Registered Authors
- Yunsheng, Zhang
- Keywords
- Gal4/UAS, Methylation, Toxicity, Zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified/genetics*
- DNA-Binding Proteins/genetics*
- DNA-Binding Proteins/metabolism*
- Molecular Biology/methods*
- Recombinant Proteins/genetics
- Recombinant Proteins/metabolism
- Regulatory Sequences, Nucleic Acid*
- Saccharomyces cerevisiae Proteins/genetics*
- Saccharomyces cerevisiae Proteins/metabolism*
- Staining and Labeling/methods
- Transcription Factors/genetics*
- Transcription Factors/metabolism*
- Zebrafish/genetics*
- PubMed
- 30613898 Full text @ Appl. Microbiol. Biotechnol.
Citation
Zhang, Y., Ouyang, J., Qie, J., Zhang, G., Liu, L., Yang, P. (2019) Optimization of the Gal4/UAS transgenic tools in zebrafish. Applied Microbiology and Biotechnology. 103(4):1789-1799.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping