A dioxin response element in the multiple cloning site of the pGL3 luciferase reporter influences transcriptional activity.
Luciferase reporter plasmids (pGL3 backbone, Promega) have been utilized to characterize the transcriptional effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands. Following ligand activation, the AhR and its dimerization partner AhR nuclear translocator (ARNT) regulate transcription by binding dioxin response elements (DREs) in regulatory regions of dioxin-sensitive genes. Upon sequencing of our luciferase reporters, we unexpectedly identified a DRE core motif within the multiple cloning site (mcsDRE) of the pGL3 luciferase plasmid backbone in a subset of our reporters. Therefore, the objective of this study was to determine if the mcsDRE inadvertently influences reporter activity. Utilizing deletional analysis we determined that the mcsDRE did significantly alter the transcriptional effect induced by TCDD. Since many chemicals have been shown to interact with the AhR and influence transcription through the DRE, the presence of the mcsDRE in the pGL3 luciferase plasmid may inappropriately influence promoter and enhancer analysis. As such, insertion of regulatory elements into pGL3 reporters should be designed to avoid retaining the mcsDRE core motif (GCGTG) and currently utilized pGL3 reporters should be evaluated for the presence of the mcsDRE.
Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Sharon D
SD
Ochs
Jing
J
Liu
Tharu M
TM
Fernando
Roger A
RA
Fecher
Courtney E W
CE
Sulentic
D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004147:Dioxins / Q000633:toxicity*; D017930:Genes, Reporter / Q000235:genetics*; D008156:Luciferases / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D020218:Response Elements / Q000235:genetics*; D014158:Transcription, Genetic
D004147:Dioxins; D008156:Luciferases