Stable Expression of T7 RNA Polymerase Gene Mediated by Retroviral Vector in PK15 and SK6 Cells

The experiment was conducted to insert T7 RNA polymerase(RNAP) gene into cells of swine by retroviral vector system,then to analyze its hereditary stability.The bacteriophage T7 RNAP gene was amplified via PCR from lysogen DE3,and the gene was cloned into pBABEpuro retrovial vector to construct a recombinant plasmid that named as pBABEpuro/T7.Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by Lipfection 2000,some pseudotype viruses were ingathered and transfected into PK15 and SK6 cells under polybrene,respectively.The T7-PK15 and T7-SK6 cells were propagated in DMEM with puromycin respectively.The genome extraction from the cells transfected different times,and the T7 RNAP gene was amplified from the genome by PCR.Expression and activity of T7 RNAP gene in T7-PK15 and T7-SK6 cells were analyzed by immediate fluorescence and SDS-PAGE assay.Results showed that the T7 RNAP gene had been integrated into the chromosome of T7-PK15 and T7-SK6 cells,and expressed stably at high level.After 40 times passage,transcriptional activity of T7-PK15 and T7-SK6 cell lines were not weaked.T7 RNAP gene had been stably integrated into the chromosome of T7-PK15 and T7-SK6 cells,construction of cell lines provided favorable tools for virus rescue in vivo.