Abstract
Kisspeptin is a multifunctional peptide encoded by the Kiss1 gene that plays critical roles in mammalian puberty onset modulation and fertility maintenance in the hypothalamus. Understanding how Kiss1 expression is regulated is essential for elucidating the molecular mechanisms responsible for these reproductive events. In this study, we constructed an in vitro dual fluorescence reporter system to facilitate high throughput screening of effectors influencing the expression of Kiss1. In GT1‐7 cells, an enhanced GFP gene was placed under the control of the Kiss1 gene regulatory elements and translated together with this gene. A tdTomato gene cassette was simultaneously introduced into the same cell for normalization of the fluorescence signal. After treatment with different effectors, the cells were analyzed by flow cytometry. We first tested the efficacy of the system using canonical regulators and then carried out high throughput functional screening to identify chemical compounds that can regulate Kiss1 gene expression. Of 22 tested compounds from natural sources, 13 significantly affected Kiss1 expression. Verification by western blot and quantitative reverse transcription PCR (qRT‐PCR) assays and structural analysis identified two chalcone compounds as possible regulators of Kiss1 gene expression. This system may be suitable for gene functional analysis, drug screening and pharmaceutical studies.
Highlights
We construct a dual fluorescence reporter system (DFRS) to monitor the expression of the Kiss1 gene, with the green fluorescence intensity of enhanced GFP (eGFP) representing the activity of transcription and translation of Kiss1, and the red fluorescence intensity of tdTomoto normalizing the non-specific factors affecting the protein synthesis in the cell
Through homology directed repair-mediated targeting with the help of CRISPR/Cas9 technology, an eGFP gene was inserted into the open reading frame of the Kiss1 gene, and transcribed and translated together with the Kiss1 gene, regulated by the full set of regulatory elements of the Kiss1 gene
Kiss1 expression can be visualized with a fluorescence detector, which made possible rapid and high throughput screening for effectors affecting Kiss1 expression
Summary
A dual fluorescence reporter system for high throughput screening of effectors of Kiss gene expression. Keywords dual fluorescence reporter system; expression effector screening; flow cytometry; high throughput; Kiss gene; kisspeptin. Verification by western blot and quantitative reverse transcription PCR (qRT-PCR) assays and structural analysis identified two chalcone compounds as possible regulators of Kiss gene expression. This system may be suitable for gene functional analysis, drug screening and pharmaceutical studies. We constructed an in vitro dual fluorescence reporter system (DFRS) to facilitate the high throughput screening of effectors that influenced Kiss gene expression. Quantitative reverse transcription PCR (qRT-PCR) assay and structural analysis, two chalcone compounds were deemed to be potential regulators of Kiss gene expression
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