Semliki Forest virus-based DNA expression vector: transient protein production followed by cell death.
We have constructed a novel DNA expression vector based on Semliki Forest virus (SFV). SFV produces non-structural proteins (nsPs) which replicate genomic RNA and amplify the mRNA encoding the structural proteins of SFV. A recombinant cDNA genome of SFV, in which the SFV structural genes were replaced by a polylinker cassette to allow for insertion of heterologous DNA, was placed under the control of a cytomegalovirus immediate-early enhancer/promoter with a polyadenylation signal. Transfection of mammalian cells with this SFV-based plasmid vector, pSFV3-CMV-lacZ-pA, resulted in transient high-level expression of a beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSFV3-CMVlacZ-pA was more than 20-fold higher than that obtained from the plasmid with deleted nsPs genes, pSFV3A5976-lacZ, demonstrating that the nsPs genes were essential for the high level of expression. Substantial beta-galactosidase activity was detected in the medium of pSFV3-CMV-lacZ-pA-transfected cells, suggesting that the overproduction of beta-galactosidase caused cell death and release of the protein into the medium. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from pSFV3-CMV-lacZ-pA constructed using an SFV replication system.
Kohno
A
A
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Emi
N
N
Kasai
M
M
Tanimoto
M
M
Saito
H
H
D000818:Animals; D018929:Cell Culture Techniques; D016923:Cell Death / Q000235:genetics; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D012672:Semliki forest virus / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D017361:Viral Nonstructural Proteins; D001616:beta-Galactosidase