RESUMEN
Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.
Asunto(s)
Células Clonales/metabolismo , Expresión Génica , Genes Reporteros , Lactamas , Proteínas Nucleares , Transcripción Genética , beta-Lactamasas/genética , Animales , Línea Celular , Separación Celular/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Transferencia de Energía , Citometría de Flujo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Semivida , Humanos , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Factores de Transcripción NFATC , Sensibilidad y Especificidad , Espectrometría de Fluorescencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Umbeliferonas/metabolismo , beta-Lactamasas/metabolismo , beta-Lactamas/metabolismoRESUMEN
Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.