A Stable Luciferase Reporter System to Characterize LXR Regulation by Oxysterols and Novel Ligands

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1951; p. 15
• Main Authors: Hutchinson, Samantha A, Thorne, James L
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: Data Analysis; Gene Dosage; Gene Expression Regulation - drug effects; Genes, Reporter; Genetic Vectors - genetics; Humans; Ligands; Liver X Receptors - genetics; Liver X Receptors - metabolism; Oxysterols - pharmacology; Transduction, Genetic; Liver X receptor; Luciferase reporter assay; Nuclear receptors; Oxysterols
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9130-3_2

Nuclear receptors (NRs) are ligand-activated transcription factors. Class 2 NRs, such as the liver X receptor (LXR)α and (LXR)β, are typically retained in the nucleus bound to the DNA in both the presence and absence of ligand. Upon binding ligands including hydroxylated cholesterol, LXR releases corepressor proteins in exchange for coactivators resulting in target gene transcription. Activity of the LXRs therefore depends on a combination of the local ligand concentration(s) and cofactor expression, which itself is a function of cell and tissue type, mutation load, and epigenetic regulation. Cross talk with other transcription factors or signaling pathways can also alter LXR activity. The role that LXR plays in both normal physiology and disease progression is becoming increasingly apparent, and a better understanding of how and when LXR is activated or repressed is pressing biological and clinical questions.The complexity of LXR regulation makes identifying novel ligands and determining LXR activity in new cell types challenging. Generating cell lines that contain a stably integrated luciferase reporter gene with an upstream LXR-dependent promoter provides a quick, cheap, robust, efficient, and high-throughput solution to identify novel ligands and assess ligand activity in new cell types. Transplant of these stable cell culture cell lines as xenografts allows reporter activation to be assessed in vivo. Here we describe the generation of stable LXR reporter cell lines, how to confirm transgene insertion and select single cell clones, as well a method to assess transgene activity in vitro.