Comparison of conventional plasmid vector and Semliki forest virus-derived vectors in expressing growth hormone releasing hormone (GHRH)

The elements for the Semliki forest virus (SFV) RNA replicon were obtained from the Alphavirus genome. It was designed to overcome the poor efficacy of some current plasmid vectors. Genes coding for viral replicases are preserved while genes coding for structural proteins are replaced by foreign genes in the RNA replicon. High levels of RNA replication and expression of foreign genes in the cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the improvement of gene expression, a LacZ gene was inserted into pIRES-neo digested by BamHI and dephosphorylated by alkaline phosphatase to construct pIRES-neo-LacZ. The RNA replicon vector pCMV-rep-LacZ and two conventional cytomegalovirus (CMV) promoter-based vectors (pLNCX-LacZ and pIRES-neo-LacZ) were transfected, using Lipofectin, to prepared 293 cells. Growth hormone releasing hormone (GHRH) expression vectors (pCMV-Rep-GHRH, pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were also tested using the same procedure. Target gene expression was detected with radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the expression level of the RNA replicon vector was 2–3 times higher than with normal plasmid vectors. This result will help to improve the efficiency of gene expression in eukaryotic cells.