Efficient selection for high-expression transfectants with a novel eukaryotic vector.

来自 dx.doi.org

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作者：

N Hitoshi，Y Ken-Ichi，M Jun-Ichi

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摘要：

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 β-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 μg/ml) effectively select for transfectants containing a high vector copy number (> 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.

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关键词：

Recombinant DNA β-actin promoter bovine papilloma virus transfection interleukin-2 G418 selection copy number Ac, gene encoding β-actin AG, modified chicken β-actin promoter bp, base pair(s) βGal, β-galactosidase BPV, bovine papilloma virus CAG, AG promoter with CMV-IE enhancer CHO, Chinese hamster ovary CMV, cytomegalovirus dhfr, gene encoding dihydrofolate reductase HMW, high molecular weight HSV, Herpes simplex virus IE, immediate early IL-2, interleukin-2 kb, kilobase(s) or 1000 bp LTR, long terminal repeat MEM, modified Eagle's medium neo, gene (NmR) encoding NPT II Nm, neomycin NPT II, Nm phosphotransferase II nt, nucleotide(s) ori, origin of DNA replication R, resistance RSV, Rous sarcoma virus SV40, simian virus 40 tk, gene encoding thymidine kinase u, unit(s)