Establishment Of A ST Cell Line Stably Expressing T7 RNA Polymerase

ST cell line, a well-established cell line derived from swine testicles, is among several cell types permissive to CSFV (including most wild-type and several vaccine strains). ST cells are also susceptive to some other swine viruses. Thus ST cells are usually used to isolate and propagate these viruses. This study was aimed to establish a ST-based cell line stably expressing the T7 RNA polymerase, which can be used in the reverse genetic manipulation of CSFV and other swine RNA viruses. To this end, a tri-cistron expression plasmid containing T7 RNA polymerase gene, enhanced green fluorescent protein (EGFP) gene, and neomycin resistance gene was constructed. Co-transfection of ST (swine testicle) cells with the recombinant resulting plasmid and another recombinant plasmid containing red fluorescent protein (RED) gene under the control of T7 promoter resulted in efficient RED expression. A stable cell line, designated as ST/T7, was screened from ST cells transfected with the tri-cistron expression plasmid with the aid of the EGFP reporter and G418 resistance, and shown to express T7 RNA polymerase efficiently and stably, as demonstrated by transient transfection of the stable cell line of different passages with the RED gene-containing plasmid. The stable expression cell line can be used to rescue virus from cloned cDNA of some porcine RNA viruses.