Efficient Transfection of VeroE6 Cells with DNA andRNA for Luciferase Reporter Assays Using FuGENE R HDTransfection Reagent

摘要

The interferon (IEN) system of mammalian cells is the firstline of defense against viral infections. Following infec-tion, danger signals such as double-stranded RNA orsingle-stranded RNA with an unmodified 5" triphosphateend (e.g., VSV-RNA) are recognized by membrane-boundToll-like receptors and/or cytoplasmic receptors (MDA5,RIG-I). This initiates a signal cascade which leads to theproduction of type I interferons. By autocrine and para-crine stimulation via the IFN-a/(3 receptor the expressionof antiviral proteins is induced in the infected cell as wellas in the surrounding cells. Many viruses express so-called interferon antagonists to subvert this system inorder to infect their target cells successfully [1]. A ver-satile tool to investigate whether a viral gene producthas IFN-antagonistic functions is the luciferase reporterassay, which measures the influence of the viral proteinon luciferase expression driven by promoters specific forthe IFN system (e.g., the human IFN-f3 promoter). When activation of the promoter is blocked in the presence ofthe viral protein, no increase in luciferase activity will beobserved, which may indicate an antagonistic function.However, non-specific effects on luciferase expression,such as cytotoxicity caused by the viral protein or thetransfection reagent, must be excluded to avoid wronginterpretation of the results. To this aim, co-expressionof a second reporter under the control of a constitutivelyactive promoter can be used to monitor cell viability andtransfection efficiency.