Aim: To check if the common used constitutively promoters, such as CMV, TK and SV40 could be responded to the nuclear factor of activated T cell (NFAT), and to explore the strategies to choose rational internal control in the dual luciferase reporter assay.
Methods: pCMV-luc vector, in which luciferase activity is driven by CMV promoter, was cloned by amplifying the CMV promoter fragment from the pCDNA3.1 vector and then inserting the CMV promoter region into the pGL3-basic vector using the standard protocol. pTK-Luc reporter was similarly constructed, with the TK promoter from the pRL-TK vector. The constructed pCMV-Luc or pTK-Luc was co-transfected with pBIND or pRL-TK respectively, together with NFAT or constitutively active form named NFATCA. Relative luciferase activity was calculated as instructed by the manual instruction.
Results: Both pCMV-Luc and pTK-Luc vectors were successfully constructed. Luciferase activity assay revealed that SV40 promoter responded to active NFAT.
Conclusion: The common used internal control promoter SV40 could respond to active NFAT, which should be kept in mind for selection of the rational internal control vector in the dual luciferase reporter assay. In addition, our study here also provides a practical strategy for rational selection of the internal control.