A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors

Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized rep orter vector primarily intended for use in reporter cell lines expressing s uch receptors, The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive sive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter con struct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase, The c himeric protein allows for both clonal selection by fluorescence, which fac ilitates the selection of optimal reporter cell lines and high-throughput s creening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters, The report er gene was more strongly inducible with increasing numbers of TRE motifs, The constructs were tested in two cell lines, CHO and HeLa, The latter regu lated reporter gene activity stronger in response to PMA (phorbol 12-myrist ate 13-acetate) stimulation and were used to construct HF1 reporter cell li nes. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha (1b) receptor. After max imal agonist stimulation reporter gene activity was increased 200-, 15-, an d 50-fold, respectively, (C) 2001 Academic Press.