Efficient selection for high-expression transfectants with a novel eukaryotic vector.
We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.
Niwa
H
H
Institute for Medical Genetics, Kumamoto University Medical School, Japan.
Yamamura
K
K
Miyazaki
J
J
D000199:Actins / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D052688:Bovine papillomavirus 1 / Q000235:genetics*; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D005822:Genetic Vectors / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D019868:Kanamycin Kinase; D007739:L Cells; D051379:Mice; D010770:Phosphotransferases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012641:Selection, Genetic; D014162:Transfection / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000199:Actins; D005839:Gentamicins; D007376:Interleukin-2; D011994:Recombinant Proteins; C010680:antibiotic G 418; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D001616:beta-Galactosidase