A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production.
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.
Kirinaka
H
H
Department of Biotechnology, School of Engineering, Nagoya University, Japan.
Kamihira
M
M
Iijima
S
S
Kobayashi
T
T
D000818:Animals; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D006358:Hot Temperature; D011994:Recombinant Proteins / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D011994:Recombinant Proteins; D004921:Erythropoietin