Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.
Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.
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Ca2 转运 ATP 酶和二氢叶酸还原酶选择标记在哺乳动物细胞系统中的偶联表达。
DOI：
10.1016/0003-9861(92)90608-y
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发表时间：
1992
影响因子：
3.9
通讯作者：
Inesi,G
中科院分区：
文献类型：
--
作者：
Hussain,A;Lewis,D;Sumbilla,C;Lai,LC;Melera,PW;Inesi,G
将鸡快肌Ca ~（2+）转运ATP酶基因克隆到人β-actin基因启动子下游，并将突变型二氢叶酸还原酶基因（A3/DHFR）克隆到SV 40启动子-增强子下游的双启动子表达载体（pHβ FCaA 3）中，获得了鸡快肌Ca ~（2+）转运ATP酶全长cDNA在中国仓鼠肺细胞系DC-3F中的稳定表达。由于其基本上正常的催化活性和适度（20倍）的抗叶酸剂甲氨蝶呤（MTX）的抗性，A3/DHFR突变酶作为一个有效的显性选择标记，在转染的细胞群体挑战与MTX，并在广泛的药物浓度范围内，允许随后的扩增和载体序列的过表达。在稳定的转染子中，表达的ATP酶被靶向细胞内膜，并且来自这些细胞的微粒体组分表现出高速率的Ca 2+转运。在使用COS 1细胞中瞬时表达的比较实验中，每个转染细胞的ATP酶水平更高，但不到5%的转染群体表现出ATP酶表达。此外，与稳定系相反，瞬时表达细胞不能繁殖。总体而言，在稳定和瞬时表达系统中，ATP酶的产量分别为12-16和4-6 μg/mg微粒体蛋白。因此，稳定转染的细胞系的优点在于ATP酶表达的均一性及其在细胞和微粒体中的分布，在于通过连续细胞增殖获得的微粒体的高产率，以及在于微粒体的可再现的功能特征。此外，来自稳定转染的细胞系的微粒体提供了一个方便的系统，用于Ca 2+转运和ATP酶部分反应的研究，消除了进行重复瞬时转染以获得足够量的酶用于功能研究的需要。
Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pHβFCaA3) in which the ATPase was cloned downstream of a human β-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12–16 and 4–6 μg per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+transport and ATPase partial reactions, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.