A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

Abstract

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neor gene. Replication of this plasmid was repressed above 39 degrees C and vigorous DNA propagation was observed below 33 degrees C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature 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 production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neor gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37 degrees C.