A coumermycin/novobiocin-regulated gene expression system
A coumermycin/novobiocin-regulated gene expression system
复制标题
DOI：
10.1089/104303403322542266
复制
发表时间：
2003-11-01
影响因子：
4.2
通讯作者：
Shen, SH
中科院分区：
文献类型：
--
作者：
Zhao, HF;Boyd, J;Shen, SH
转基因在哺乳动物中的调控表达是功能基因组研究和临床应用的重要技术。在这里，我们描述了哺乳动物的基因表达调控系统，基于香豆素开关的细菌DNA促旋酶B亚基（GyrB）的二聚化。通过将GyrB激活因子融合到细菌λ阻遏物结合结构域来构建反式激活因子。纳摩尔浓度的抗生素香豆霉素通过同二聚化嵌合反式激活因子与位于微型启动子上游的λ操纵子的结合来激活转基因。更重要的是，新生霉素，香豆霉素的拮抗剂，通过废除香豆霉素诱导的反式激活因子的二聚化，迅速关闭了基因的表达。λ阻遏物结合结构域的定点诱变导致基础表达水平的显著降低，并且在稳定转染的293 A细胞中响应于香豆霉素的诱导达到四个数量级。通过在哺乳动物细胞中诱导表达促凋亡bax基因的稳定细胞系的制备证明了这种诱导系统用于严格调控基因表达的能力。因此，这种新的香豆素开关/开关系统应扩大调节基因表达的效用，特别是当需要快速开/关交换。
Regulated expression of transgenes in mammals is an important technique in both functional genomic studies and clinical applications. Here we describe a regulated gene expression system for mammals, based on coumarin-switched dimerization of the bacterial DNA gyrase B subunit (GyrB). The transactivator was constructed by fusing the GyrB activator to the bacterial lambda repressor-binding domain. The antibiotic coumermycin in nanomolar concentrations activated the transgene through binding of the homodimerized chimeric transactivator to the lambda operator located upstream of a minipromoter. More significantly, addition of novobiocin, an antagonist of coumermycin, promptly switched off expression of the gene by abolishing coumermycin-induced dimerization of the transactivator. Site-directed mutagenesis of the lambda repressor-binding domain resulted in significant reduction of basal expression levels and an induction reaching four orders of magnitude in stably transfected 293A cells in response to coumermycin. The capability of this inducible system for tightly regulated gene expression was demonstrated by the ready generation of stable cell lines inducibly expressing the proapoptotic bax gene in mammalian cells. Hence, this novel coumarin switch-on/switch-off system should broaden the utility of regulated gene expression, particularly when rapid on/off interchange is required.