Transient gene expression in mammalian and mosquito cells using a recombinant Semliki Forest virus expressing T7 RNA polymerase.
In this report, we describe a novel recombinant Semliki Forest virus (SFV) expressing T7 RNA polymerase (T7-RP), which was shown to drive transient expression of the chloramphenicol acetyltransferase (cat) gene in mammalian and mosquito cells after transfection of plasmids carrying the reporter gene under the control of the T7 promoter. To our knowledge, this is the first description of a T7-RP-based expression in mosquito cells. Expression of the cat gene was significantly enhanced in mammalian cells by inserting the sequence of the encephalomyocarditis virus internal ribosome entry site between the T7 promoter and the 5' end of the cat gene. In mosquito cells, the level of chloramphenicol acetyltransferase activity was increased by flanking the cat gene with the Autographa californica baculovirus p10 gene 5' and 3' untranslated regions. SFV expressing T7-RP appears, therefore, to be an alternative to other virus-based gene-expression systems in mammalian cells and a powerful tool for protein expression in mosquito cells.
Kohl
A
A
Unité des Arbovirus et virus des fièvres hémorragiques Institut Pasteur, Paris, France. kohla@pasteur.fr
Billecocq
A
A
Préhaud
C
C
Yadani
F Z
FZ
Bouloy
M
M
D000330:Aedes; D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015870:Gene Expression; D012672:Semliki forest virus / Q000235:genetics*; D014764:Viral Proteins
D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases