EFFICIENT SELECTION FOR HIGH-EXPRESSION TRANSFECTANTS WITH A NOVEL EUKARYOTIC VECTOR
EFFICIENT SELECTION FOR HIGH-EXPRESSION TRANSFECTANTS WITH A NOVEL EUKARYOTIC VECTOR
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DOI：
10.1016/0378-1119(91)90434-d
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发表时间：
1991-12-15
期刊：
影响因子：
3.5
通讯作者：
MIYAZAKI, J
中科院分区：
文献类型：
--
作者：
NIWA, H;YAMAMURA, K;MIYAZAKI, J
我们开发了一种新的表达载体，可以有效地选择高水平表达外源基因的转染子。该载体由一个基于 β-肌动蛋白启动子的普遍强启动子、牛乳头状瘤病毒基因组 69% 的亚区域和一个由弱启动子驱动的突变新霉素磷酸转移酶 II 编码基因组成，该启动子仅赋予对 G418 的边际抗性。因此，高浓度的 G418（约 800-mu-g/ml）可以有效地选择含有高载体拷贝数（> 300）的转染子。我们通过在 L 细胞和中国仓鼠卵巢 (CHO) 细胞中产生人白细胞介素 2 (IL-2) 来测试该系统，结果表明，高浓度的 G418 有效地产生了 L 细胞和 CHO 细胞转染子，其稳定产生 IL-2 的水平与之前使用基因扩增获得的水平相当。发现载体序列已整合到宿主染色体中，并在转染子中稳定维持几个月。
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-mu-g/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.