Controlled gene expression in mammalian cells via a regulatory cascade involving the tetracycline transactivator and lac repressor
Controlled gene expression in mammalian cells via a regulatory cascade involving the tetracycline transactivator and lac repressor
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DOI：
10.1016/0378-1119(96)00216-8
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发表时间：
1996-06-26
期刊：
影响因子：
3.5
通讯作者：
Schiestl, RH
中科院分区：
文献类型：
--
作者：
Aubrecht, J;Manivasakam, P;Schiestl, RH
调节级联或调节通过一个初始信号在多个点或多个基因上控制通路。在本文中，我们描述了CHO细胞中人工调节级联的构建，该级联对不同浓度的四环素（Tc）和/或IPTG作出反应。该系统由组成性产生的Tc操纵子（tet）的反激活子（TTA）组成，其诱导由tet操作子（tetO）和上游CMV启动子（p*CMV）序列控制的lad基因的表达。LacI通过结合上游RSV启动子（pRSV）区域的lacO位点来抑制cat基因的活性。然而，这种抑制可以通过暴露于Tc或IPTG来缓解，它们分别抑制了TTA和LacI的结合活性。因此，用Tc或IPTG治疗导致CAT活性增加10倍。诱导剂停用后，cat表达恢复到基础水平。Tc的调节表现出表型滞后，在192 h后达到完全诱导，而加入IPTG后在24 h内达到完全诱导。当Tc和IPTG同时处理细胞时，在24 h内达到完全诱导，并且在Tc单独存在时保持完全诱导。这表明Tc的调控是快速的，表型滞后可能是由于LacI抑制因子的缓慢周转。这个TTA/lacI调控系统可以作为cat表达作为报告者的一个例子。这些数据表明，在哺乳动物细胞中，任何克隆基因都可以构建多点调控的级联。
Regulatory cascades or regulons control pathways at multiple points or multiple genes by one initial signal. In this paper, we describe the construction of an artificial regulatory cascade in CHO cells, which responded to various concentrations of tetracycline (Tc) and/or IPTG. The system consists of the constitutively produced transactivator (TTA) of the Tc operon (tet), which induced the expression of a lad gene controlled by tet operator (tetO) and upstream CMV promoter (p*CMV) sequences. LacI repressed the activity of a cat gene by binding to lacO sites in its upstream RSV promoter (pRSV) region. However, this repression could be alleviated by exposure to Tc or IPTG, which inhibited the binding activities of TTA and LacI, respectively. Hence, treatment with either Tc or IPTG led to a tenfold increase in CAT activity. After the withdrawal of the inducer, cat expression reverted to basal levels. Regulation by Tc showed a phenotypic lag, and full induction was reached after 192 h, whereas IPTG addition led to full induction within 24 h. When cells were treated with both Tc and IPTG, full induction of cat was reached in 24 h and maintained thereafter while in the presence of Tc alone. This suggests that regulation by Tc is fast and that the phenotypic lag may be due to slow turnover of the LacI repressor. This TTA/lacI regulatory system may serve as an example in which cat expression was used as a reporter. The data indicate that regulatory cascades regulated at multiple points can be constructed with any cloned gene in mammalian cells.