Abstract
Bi-directional promotion has been reported in various species. Understanding transcriptional control at these various loci requires a tool which measures bi-directional transcription. Green Fluorescent Protein (GFP) reporters may be suitable for simultaneous measurement in a dualreporter system. GFP variants have similar half-lives, sensitivity for detection, and distinct excitation and emission wavelengths. The GFP variant, ECFP and EYFP, were chosen as reporter genes reconstructed into the same plasmid in a head-to-head orientation. Each reporter protein emitted either cyan or yellowish-green fluorescence at distinct excitation and emission wavelengths. In this study, dual-promoter dual-reporter vectors based on GFP variants were developed and tested using an artificial bidirectional promoter constructed from the SV40 and TK minimal promoters. For up to 44 hours after transfection, the intensity of the ECFP and EYFP reporters directed by the TK promoter correlated with the amount of plasmid being transfected and remained in the linear range. The background autofluorescence of Hep G2 cells was low and remained stable during the time frame of the experiments. The reorientation of the reporter gene or insertion of a reoriented reporter and its promoter did not significantly affect the background fluorescence of a promoterless GFP reporter in the same plasmid. Also, the expression of the reporter gene was not significantly affected by the presence of a promoterless reporter in the reverse orientation. The dual-promoter dual-reporter vectors were evaluated and proved to be capable of expressing dual-reporter genes controlled by dual-promoters. These dual-reporter plasmids could be used to determine the presence of bidirectional promotion and should allow deletion analysis for the localization of minimal promoter elements. A retinoic acid-responsive element, DR5, was inserted between the SV 40 and TK promoters to test whether this dualreporter vector could be used in characterizing a cA-acting enhancer within this bi-directional promoter region. Reporter expression was minimally induced in retinoic acid (RA)-treated cells. These studies demonstrated that plasmids based on GFP variant reporter genes could be adapted to the study of bi-directional promotion.