Establishment of a PK-15 cell line stably expressing T7 RNA polymerase

To establish a PK-15 cell line stably expressing T7 RNA polymerase (T7 RNAP), the T7 RNAP gene was amplified by PCR from E. coli BL21 (DE3) cells and inserted into the retroviral plasmid vector pLXSN to construct the recombinant plasmid pLXSN-T7. The pLXSN-T7 was transfected into PT67 cells to prepare the recombinant retrovirus (rMLV-T7) in the presence of G418. The PK-15 cells stably expressing the T7 RNAP. designated PK/T7, was established by rMLV-T7 infection, G418 selection and immunofluorescence assay. T7 RNAP transcription activity was detected in the PK/T7 cells via transfection with pET-RED for transient expression of red fluorescent protein. These results indicated that the PK/T7 cells could be an useful tool for reverse genetic manipulation of porcine RNA viruses.