A reporter system for reverse transfection cell arrays.
The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conventional 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 technology 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.
Webb
Brian L
BL
Science and Technology Division, Corning Incorporated, Corning, NY 14832, USA. webbbl@corning.com
Díaz
Begoña
B
Martin
G Steven
GS
Lai
Fang
F
D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D002477:Cells / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D015336:Molecular Probe Techniques; D009570:Nitriles / Q000494:pharmacology; D027602:Serum Response Element / Q000235:genetics; D014162:Transfection
D002070:Butadienes; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009570:Nitriles; C113580:U 0126; D049452:Green Fluorescent Proteins; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases