2. Academic Validation
  3. CYP26A1 gene promoter is a useful tool for reporting RAR-mediated retinoid activity

CYP26A1 gene promoter is a useful tool for reporting RAR-mediated retinoid activity

  • Anal Biochem. 2019 Jul 15:577:98-109. doi: 10.1016/j.ab.2019.04.022.
Reza Zolfaghari 1,  Floyd J Mattie 2,  Cheng-Hsin Wei 2,  David R Chisholm 3,  Andrew Whiting 3,  A Catharine Ross 4
Affiliations

Affiliations

  • 1 Department of Nutritional Sciences, Pennsylvania State University, University Park, PA, USA. Electronic address: rxz7@psu.edu.
  • 2 Department of Nutritional Sciences, Pennsylvania State University, University Park, PA, USA.
  • 3 Centre for Sustainable Chemical Processes, Department of Chemistry, Science Laboratories, Durham University, South Road Durham, DH1 3LE, UK.
  • 4 Department of Nutritional Sciences, Pennsylvania State University, University Park, PA, USA; Center for Molecular Immunology and Infectious Disease, Pennsylvania State University, University Park, PA, USA.
PMID: 31039331 DOI: 10.1016/j.ab.2019.04.022
Abstract

Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and Other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.

Keywords

CYP26A1 gene promoter; Luciferase assay; Red fluorescent protein; Retinoic acid; Retinol.

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