Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.
Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005784:Gene Amplification; D005814:Genes, Viral; D009975:Orthomyxoviridae / Q000235:genetics*; D010957:Plasmids; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D004262:DNA Restriction Enzymes