Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines.

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site (IRES) sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells.For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity.Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient.Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics.