Abstract
BackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish.ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.ConclusionOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.
Highlights
Zebrafish are a foundational model organism for studying the spatiotemporal activity of genes and their regulatory sequences
The CMV enhancer is frequently used in reporter vector construction across a wide range of studies due to its robust and constitutive eGFP expression (63/69 GFP+ at 24 h, 49/49 GFP+ at 48 h) (Supplementary Figure 1B)
The lac Operator-Repressor System Is Functional in the PAC2 Zebrafish Cell Line
Summary
Zebrafish are a foundational model organism for studying the spatiotemporal activity of genes and their regulatory sequences. Experimental approaches for the study of transcriptional regulation by cis-regulatory elements in vivo require methods for both genetically modifying cells or organisms, and for measuring expression levels of specific genes. Zebrafish (Danio rerio) is an ideal model organism for investigating the spatio-temporal-specific regulation of gene expression throughout the developing embryo as it satisfies the requirements for ease of genetic manipulation and expression readout. Due to its benefits as a model organism, many technologies for studying gene function have been developed in zebrafish, including Cre/lox (Langenau et al, 2005), tamoxifen-inducible Cre (Hans et al, 2009), Inducible lac Operator-Repressor in Pac the Tet-On system (Campbell et al, 2012), RNAi (Dodd et al, 2004; Kelly and Hurlstone, 2011), and more recently, CRISPR based-methods (Hwang et al, 2013). The use of the lac operator-repressor system, a tool that functions transiently in a native context with minimal disruption of local regulation compared to many of the aforementioned methods, has yet to be demonstrated in zebrafish
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