ABSTRACTUnderstanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. In this study, inhibitory regions in the 5’ end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. This finding was reached in the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modification were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were restored. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines. This study showed that inserting 261 to 313 bp from the 3’ end of the OpIE2 promoter downstream of the CMV promoter maintains efficient gene expression in both Sf9 and mammalian cells.
The baculovirus promoter OpIE2 sequence has inhibitory effect on the activity of the Cytomegalovirus (CMV) promoter in HeLa and HEK-293T cells
