Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression.
Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression.
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DOI：
10.3791/50171
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发表时间：
2013-03-05
期刊：
影响因子：
--
通讯作者：
Hergovich A
中科院分区：
文献类型：
--
作者：
Gomez-Martinez M;Schmitz D;Hergovich A
哺乳动物细胞生物学领域的一个主要方法是在选定的细胞系中操纵感兴趣基因的表达，目的是通过瞬时/稳定的过表达或敲除感兴趣基因来揭示该基因的一个或多个功能(S)。不幸的是，当基因表达的操纵导致细胞生长/增殖缺陷或不想要的细胞分化时，这种方法不适合于各种细胞生物学研究。因此，研究人员已经改造了从大肠杆菌四环素耐药操纵子中提取的四环素抑制蛋白(TetR)，以产生非常高效和严格的调控系统，以在哺乳动物细胞中表达cDNA。简而言之，TetR已被修饰为：(1)加入四环素后通过与启动子区域的Tet-操纵子(TO)结合来阻止转录的启动(称为Tet-Off系统)或(2)在没有四环素的情况下与TO结合(称为Tet-On系统)(图1)。鉴于Tet-On系统需要四环素(在组织细胞培养介质中的半衰期约为24小时)的持续存在带来的不便，Tet-On系统已得到更广泛的优化，导致开发出非常紧密和高效的表达载体系统，如本文所用。在哺乳动物细胞中建立了基因敲除的RNA干扰(RNAi)后不久，表达短发夹状RNAs(ShRNAs)的载体被描述为功能非常类似于siRNAs。然而，这些shRNA介导的敲除方法与传统的敲除策略具有相同的局限性，因为当基因靶点对细胞生存至关重要时，稳定的敲除是不可行的。为了克服这一限制，van de Wetering等人。通过在启动子区域插入TO来修饰shRNA表达载体pSUPER，这使它们能够产生稳定的细胞系，其目的基因可被四环素诱导缺失。在这里，我们描述了一种有效地产生稳定的人类Tet-on细胞系的方法，该细胞系可靠地驱动可诱导的目的基因的过度表达或缺失。利用这种方法，我们成功地建立了Tet-on细胞系，这极大地促进了对中心体和凋亡信号中MST/hMOB/NDR级联信号的分析。在这份报告中，除了描述高效产生人Tet-on细胞系所必需的两个连续的操作步骤外，我们还描述了我们选择的载体(图2)。此外，除了概述人类Tet-on细胞系的产生方案外，我们还将讨论有关Tet-on细胞的技术程序和特征的关键方面。
A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene’s function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hours in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here. Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al. modified the shRNA expression vector pSUPER by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest. Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome and apoptosis signaling. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.
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影响因子：
5.3
作者：
Kohler, Reto S.;Schmitz, Debora;Hergovich, Alexander
通讯作者：
Hergovich, Alexander
影响因子：
9.2
作者：
Vichalkovski, Anton;Gresko, Ekaterina;Hemmings, Brian A.
通讯作者：
Hemmings, Brian A.
影响因子：
7.7
作者：
van de Wetering, M;Oving, I;Clevers, H
通讯作者：
Clevers, H
影响因子：
14.9
作者：
POSTLE, K;NGUYEN, TT;BERTRAND, KP
通讯作者：
BERTRAND, KP
影响因子：
56.9
作者：
Brummelkamp, TR;Bernards, R;Agami, R
通讯作者：
Agami, R