Nucleic Acids Research, 1985, Vol. 13, No. 22 7959-7977
© 1985
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A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NSI gene expression
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma Canto Blanco, 28049 Madrid +Centro Nactonal de Microbiología, Virología e Inmunología Sanitarias Majadahonda, Madrid, Spain
*To whom correspondence should be addressed
Received September 27, 1985. Accepted October 28, 1985.
Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLTs1, and followed by the SV40 late promoter contained in pSLts1, and followed buy the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.
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