A Reporter System for Reverse Transfection Cell Arrays

Abstract

The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conven tional gene discovery approaches in the pharmaceutical industry. High-throughput approaches for studying gene function in vivo are greatly needed. One potential answer to this challenge is reverse transfection, a high-throughput gene expression method for examining the function of hundreds to thousands of genes in parallel. One limitation of reverse transfection tech nology is the need for posttransfection processing of the arrays to analyze the activity of the expressed proteins. The authors have investigated the use of a reporter construct cotransfected with other genes of interest to monitor and screen gene function on reverse transfection microarrays. They developed a serum response element (SRE) reporter linked to the green fluorescent protein (GFP) that is cotransfected with target genes on reverse transfection arrays for monitoring mitogen-activated protein (MAP) kinase signaling by multiple targets in parallel. The authors show that this reporter system is able to detect inhibition of upstream MAP kinase signaling proteins by the MEK inhibitor U0126. The ability to monitor the activity of multiple signaling proteins in a multiwell format suggests the utility of reverse transfection reporter arrays for high-throughput screening applications.