Inhibitory effects associated with use of modified Photinus pyralis and Renilla reniformis luciferase vectors in dual reporter assays and implications for analysis of ISGs.
Luciferase reporter constructs are widely used for analysis of gene regulation when characterizing promoter and enhancer elements. We report that the recently developed codon-modified Renilla luciferase construct included as an internal standard for cotransfection must be used with great caution with respect to the amount of DNA transfected. Also, the dual-luciferase reporter vectors encoding Photinus pyralis firefly or Renilla reniformis luciferase showed a linear increase in dose-response with increasing amounts of transfected DNA, but at higher levels of transfected DNA, a reduction in expressed levels of luciferase activity resulted. In addition, treatment with type I interferon (IFN) was found to significantly reduce levels of P. pyralis firefly and Renilla luciferase activity. In contrast, cells transfected with a green fluorescent protein (GFP) reporter construct showed no significant IFN-associated change. The reduction in luciferase activity resulting from IFN treatment was not due to IFN-mediated cytotoxicity, as no change in cellular propidium iodide (PI) staining was observed by flow cytometry. IFN treatment did not alter the levels of firefly luciferase activity in cell culture supernatants or the luciferase mRNA levels determined by quantitative real-time RT-PCR analysis. Based on these results, it is probable that the IFN-induced reduction in levels of luciferase activity detected in reporter assays occurs via a posttranscriptional mechanism. Thus, it is important to be aware of these complications when using luciferase reporter systems in general or for analyzing cytokine-mediated responsive regulation of target genes, particularly by the type I IFNs.
Ghazawi
Ibtisam
I
Genomics Research Centre, School of Health Science, Griffith University Gold Coast Campus, Gold Coast Mail Centre, PMB50, Queensland, 9726, Australia.
Cutler
Samuel J
SJ
Low
Pauline
P
Mellick
Albert S
AS
Ralph
Stephen J
SJ
D000818:Animals; D045744:Cell Line, Tumor; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005470:Fluorometry; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D016899:Interferon-beta / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D008156:Luciferases / Q000187:drug effects / Q000235:genetics*; D049409:Luciferases, Firefly; D049410:Luciferases, Renilla; D008545:Melanoma; D010957:Plasmids; D011419:Propidium / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D048790:Renilla / Q000201:enzymology / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D005456:Fluorescent Dyes; D016898:Interferon-alpha; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D011419:Propidium; D016899:Interferon-beta; D007371:Interferon-gamma; D008156:Luciferases; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly