Using Reporter Gene Assays to Screen and Identify Chemical Compounds that Modulate Estrogen Receptor Activity

Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor α Basic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors.