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. 2021 Jun 18:12:683394.
doi: 10.3389/fgene.2021.683394. eCollection 2021.

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells

Sierra S Nishizaki  1   2 , Torrin L McDonald  1 , Gregory A Farnum  2 , Monica J Holmes  2 , Melissa L Drexel  1 , Jessica A Switzenberg  2 , Alan P Boyle  1   2
Affiliations

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells

Sierra S Nishizaki et al. Front Genet. .

Abstract

Background: Zebrafish 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.

Results: Here 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.

Conclusion: Our 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.

Keywords: GFP; lac operator-repressor system; luciferase; reporter; zebrafish.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The CMV enhancer drives strong reporter gene expression in PAC2 cells. The CMV enhancer shows a 24-fold increase in luciferase activity in PAC2 zebrafish fibroblast cells compared to a SV40 promoter-only plasmid. Error bars represent standard deviation of 3 biological replicates. Statistical significance was determined using a Student’s two tailed t-test ∗P < 0.01. Points represent values for all 3 replicates in each condition. All plasmid components are detailed as symbols above the figure. The TSS starts where the SV40 promoter begins to slope downward.
FIGURE 2
FIGURE 2
Co-transfection of LacI-expressing modules with repressible reporter modules result in LacI-mediated repression in PAC2 cells. SV40 promoter-only driven expression of LacI shows moderate repression (40%) and CMV enhancer-driven expression of LacI shows high repression (70%) of a repressible module containing 6x LacO sites. Expression of non-functional LacI (NFLacI, frameshift mutant) shows no repression and all modules showed maximal reporter expression in the presence of 1 mm IPTG. Error bars represent standard deviation of replicates (n = 3). Points represent values for all 3 replicates in each condition. The dashed line shows the NF LacI IPTG- negative control. The TSS starts where the SV40 promoter begins to slope downward. Statistical significance was determined using a Student’s two tailed t-test ∗P < 0.001.
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