Protocol

Molecular Endocrinology

Volume 590 of the series Methods in Molecular Biology pp 177-193

Date:

Use of Reporter Genes to Study the Activity of Promoters in Ovarian Granulosa Cells

  • Jingjing L. KippAffiliated withDepartment of Biochemistry, Molecular Biology and Cell Biology, Center for Reproductive Science, Northwestern University
  • , Kelly E. MayoAffiliated withDepartments of Neurobiology & Physiology and Biochemistry, Molecular Biology and Cell Biology, Center for Reproductive Science, Northwestern University

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Abstract

Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and β-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor α (ERα)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin increases ERα promoter activity and increases functional ER activity, suggesting a reciprocal regulation between activin and estrogen signaling in the ovary. We also discuss more broadly some key considerations in the use of reporter genes and cell-based transfection assays in endocrine research.

Key words

Reporter genes luciferase β-galactosidase promoter activity estrogen estrogen receptor activin