PUBLICATION
Fluorescent reporter zebrafish line for estrogenic compound screening generated using a CRISPR/Cas9-mediated knock-in system
- Authors
- Abdelmoneim, A., Clark, C., Mukai, M.
- ID
- ZDB-PUB-191108-9
- Date
- 2019
- Source
- Toxicological sciences : an official journal of the Society of Toxicology 173(2): 336-346 (Journal)
- Registered Authors
- Keywords
- biosensors, early development, endocrine disrupting chemicals (EDCs), imaging, xenobiotic estrogens, zebrafish transgenic
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Benzhydryl Compounds/pharmacology
- CRISPR-Cas Systems*
- Embryo, Nonmammalian/drug effects
- Embryo, Nonmammalian/metabolism
- Endocrine Disruptors/pharmacology
- Environmental Monitoring/methods
- Estradiol/pharmacology
- Estrogens/metabolism*
- Estrogens/pharmacology*
- Gene Expression Regulation/drug effects
- Gene Knock-In Techniques/methods*
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism*
- Larva/metabolism
- Liver/metabolism
- Molecular Sequence Data
- Phenols/pharmacology
- RNA, Messenger/metabolism
- Vitellogenins/genetics
- Vitellogenins/metabolism
- Zebrafish/genetics
- Zebrafish/metabolism
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 31688941 Full text @ Toxicol. Sci.
- CTD
- 31688941
Citation
Abdelmoneim, A., Clark, C., Mukai, M. (2019) Fluorescent reporter zebrafish line for estrogenic compound screening generated using a CRISPR/Cas9-mediated knock-in system. Toxicological sciences : an official journal of the Society of Toxicology. 173(2):336-346.
Abstract
An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. Time- and cost-effective biological tools to detect and screen these compounds with potential high-throughput capabilities are in ever-growing demand. We generated a knock-in zebrafish transgenic line with enhanced green fluorescent protein (EGFP) driven by the regulatory region upstream of vitellogenin 1 (vtg1), a well-studied biomarker for estrogenic exposure, using CRISPR/Cas9 technology. Exposure to 17β-estradiol (E2; 0-625 nM) starting at 4-hours post-fertilization in dechorionated embryos resulted in the significant induction of hepatic EGFP with ≥5 nM E2 as early as 3-days post-fertilization. Concentration- and time-dependent increase in the percentage of hepatic EGFP-positive larvae and extent of fluorescence expression, categorized into 3 expression levels, were observed with E2 exposure. A strong correlation between the levels of EGFP mRNA, vtg1 mRNA, and EGFP fluorescence levels were detected. Image analysis of the area and intensity of hepatic EGFP fluorescence resulted in high-fidelity quantitative measures that could be used in automated screening applications. In addition, exposure to bisphenol A (0-30 μM) resulted in quantitative responses showing promise for the use of this transgenic line to assess estrogenic activity of endocrine-disrupting chemicals. These results demonstrate that this novel knock-in zebrafish reporter allows for distinct screening of in vivo estrogenic effects, endpoints of which can be used for laboratory testing of samples for estimation of possible human and environmental risks.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping