Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.
Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.
复制标题
在过表达糖皮质激素受体的 CHO 细胞中，含有多个 GRE 的载体可高度诱导表达。
DOI：
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发表时间：
1989
影响因子：
14.9
通讯作者：
Randal J. Kaufman
中科院分区：
文献类型：
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作者：
David I. Israel;Randal J. Kaufman
描述了一种条件糖皮质激素应答表达载体系统，用于在哺乳动物细胞中高度诱导表达外源基因。这种宿主-载体系统需要糖皮质激素受体（GR）蛋白在宿主细胞中的高水平表达和表达载体内受体结合位点的多个拷贝。用编码大鼠GR的表达载体转染和筛选中国仓鼠卵巢细胞，获得高水平表达功能受体的细胞系。将MMTV增强子（糖皮质激素反应元件，GRE）的多个拷贝插入到基于腺病毒主要晚期启动子（AdMLP）的表达载体中，在瞬时DNA转染试验中，地塞米松（dex）的诱导表达量大于1000倍。诱导表达量比含有SV40增强子的AdMLP载体高7倍，但缺乏GRE。含有SV40增强子和多个GRE的载体在没有dex的情况下显示出基础表达的升高，但在瞬时检测和在CHO基因组中整合和扩增后都保持了诱导性。该表达系统对于研究基因调控和异种基因的可调控表达具有普遍的实用价值。
A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion.