使用小鼠二氢叶酸还原酶的甲氨蝶呤抗性突变体转染二氢叶酸还原酶阴性（DHFR-）和二氢叶酸还原酶阳性（DHFR+）哺乳动物细胞。

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase.

作者信息

Thillet J, Pictet R

机构信息

Institut Jacques Monod, Unité INSERM 257, Université Paris VI.

出版信息

FEBS Lett. 1990 Sep 3;269(2):450-3. doi: 10.1016/0014-5793(90)81213-8.

DOI:10.1016/0014-5793(90)81213-8

PMID:2401371

Abstract

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.

摘要

采用定点诱变技术构建了小鼠二氢叶酸还原酶突变体，这些突变体对甲氨蝶呤的抗性比野生型酶更强。突变基因用于转染DHFR-或DHFR+细胞系。这些突变体以及野生型基因都能够赋予DHFR- CHO细胞对甲氨蝶呤的抗性。随着甲氨蝶呤浓度的增加，筛选出的菌落数量减少。在10微摩尔甲氨蝶呤浓度下观察到的菌落数量与该酶的Ki(MTX)相关：Ki越高，相应突变体的菌落数量越高。相比之下，转染DHFR+细胞后，野生型和突变体的菌落数量没有差异。