Establishment of a temperature-inducible cell line for human plasminogen activator (tissue-type) by transfection of monkey cells with expression constructs.
An expression construct for human tissue-type plasminogen activator (t-pA) cDNA [containing a simian virus 40 (SV40) origin of replication] was introduced into CV1, COS-7 and COSts2 cells; in the latter cell line the amount of functionally active large T antigen of SV40 is regulated by the temperature. In a transient system, the expression in COSts2 cells at the permissive temperature for large T antigen was improved sixfold compared to COS-7 cells. By cotransfection with a plasmid conferring resistance to G418 into COSts2 cells, a cell line (COSts2Glob t-pA) could be isolated with barely detectable expression of t-pA at the semi-permissive and non-permissive temperature and inducible secretion of t-pA at the permissive temperature. The kinetics of induction, inducibility after continued propagation at the semi-permissive temperature and the influence of the temperature during previous propagation on inducibility were investigated. The biological activity of the secreted material was demonstrated by a functional assay. Inducibility of t-pA by temperature was accompanied by a dramatic increase of the copy number of episomal plasmids (up to 2000 copies per cell).
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D010957:Plasmids; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D004247:DNA; D010959:Tissue Plasminogen Activator