Abstract:[Background] T7 phage-derived T7 RNA polymerase (T7 RNAP) is a commonly employed component of the reverse genetic operating system of viruses, due to its high specificity and efficient initiation of transcription. Several cell lines stably expressing T7 RNAP have been constructed for reverse genetics studies of RNA viruses. The grass carp, the freshwater fish with the highest production in China, is susceptible to grass carp reovirus (a double-stranded RNA virus). However, no grass carp cell line stably expressing T7 RNAP has been reported to date.[Objective] To construct the stable cell lines expressing T7 RNAP by using the Tol2 transposon system from grass carp fin (GCF) and grass carp ovary (GCO) cells and measure their viability, thus providing cellular tools for establishing the reverse genetic operating system for grass carp reovirus. [Methods] The T7 RNAP gene was amplified from the genomic DNA of Escherichia coli BL21(DE3) via PCR and inserted into the Tol2 transposon plasmid via homologous recombination. The recombinant Tol2 transposon plasmids carrying T7 RNAP with or without an N-terminal or C-terminal nuclear localization signal (NLS) were constructed. These recombinant plasmids, along with a helper plasmid expressing the Tol2 transposase, were co-transfected into the two grass carp cell lines, followed by selection with hygromycin B. Western blotting (WB) was employed to determine the T7 RNAP expression in the selected cells. An enhanced green fluorescent protein (EGFP) reporter plasmid driven by a T7 promoter was used to validate the functionality of T7 RNAP, and the effects of NLS on the expression and activity of T7 RNAP were studied. [Results] Three recombinant Tol2 transposon plasmids carrying the T7 RNAP gene were successfully constructed. Six stable cell lines expressing T7 RNAP were obtained after selection. WB results confirmed significant expression of T7 RNAP in all the selected cell lines. Transfection with the T7 promoter-driven EGFP plasmid demonstrated high levels of EGFP expression, confirming the transcriptional activity of T7 RNAP in these cell lines. Notably, the GCO cells expressing T7 RNAP with an NLS, especially with the C-terminal NLS, showed higher expression and transcriptional activity than those without NLS. However, no significant difference was observed in GCF cells. [Conclusion] We successfully established stable T7 RNAP-expressing GCF and GCO cell lines by using the Tol2 transposon system. These cell lines demonstrated efficient transcription and expression of genes under the control of the T7 promoter, which could be further enhanced by addition of an NLS. NLS could enhance nuclear accumulation and transcriptional efficiency of T7 RNAP by facilitating its transport into the nucleus, with the effects depending on the cell lines. The results layed a foundation for the application of T7 RNAP in the research on grass carp reovirus.
