A NEW RUNAWAY TYPE EPISOMAL VECTOR FOR MAMMALIAN-CELLS BASED AN A TEMPERATURE-SENSITIVE SIMIAN-VIRUS-40 AND INDUCIBLE ERYTHROPOIETIN PRODUCTION

A runaway vector for mammalian cells was constructed from the simian v irus 40 (SV40) genome with a temperature-sensitive mutation of the lar ge T antigen and bacterial neo(r) gene. Replication of this plasmid wa s repressed above 39(degrees)C and vigorous DNA propagation was observ ed below 33 degrees C in simian CV-1 cells. The human erythropoietin g ene was inserted downstream of the SV40 late promoter of the plasmid a nd the recombinant plasmid was introduced into CV-1 cells. By a temper ature shift from 37 to 33(degrees)C, the plasmid copy number increased from 5 x 10(2) to 5 x 10(3) copies per cell and the specific producti on rate of erythropoietin increased more than ten-fold. The bacterial derived sequences such as the neo(r) gene and vector pUC sequences wer e prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained a t either 33 or 37 degrees C.