Development of runaway vector for mammalian cells and its application for production.

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Production of biological substances by recombinant DNA technology with mammalian cells is one of the most important procedures either for basic research or industrial production. In contrast to microbial cells, plasmids have not been used as a vestor for mammalian cells. In this regard, we tried to develop a novel vector system which has big copy number and, if possible, of which the copy number could be controlled by temperature.A renaway vector for mammalian cells was constructed from simian virus 40(SV40) genome with temperature sensitive mutation of large T antigen and bacterial neo^<gamma> gene. Replication of this plasmid was repressed above 39ﾟC and vigorous DNA propagation was observed below 33ﾟC in simian CV-1 cells. Human erythropoietin was very effectively produced by using this vector. This vector worked very well in spppinner flask cultures.For industrial applications of our novel renaway-type vector for mammalian cells based on simian virus 40, we studied host range specificity of this vestor, especially with human derived cell lines, since human cells as hots seem to be very important for the production of pharmaceuticals by genetic engineering. The runaway-type vector showed a high replication rate at a permissive temperature of 33ﾟC in HeLa cells and erythropoietin was produced effectively. The plasmid copy number was dependent on the temperature but was relatively low in KB cells.We also established an episomal type expression vector for rodent cells.Cloned wild type polyoma virus genome was mutated by site-directed mutagenesis to introduce the temperature-dependent replication. The mutated polyoma was ligated wiht selection marker, and new expression vector for rodent cells, of which replication was regulated by temperature, was constructed.