筛选在瞬时转染和稳定转导的哺乳动物细胞中表达多西环素诱导基因的最佳Tet-on系统

Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells

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|核心内容：

强力霉素(Dox)诱导的Tet-on系统被广泛用于调控哺乳动物细胞中的基因表达。

该系统以细菌Tet操纵子为基础，对其在真核细胞中的功能进行了修饰和改进。

为了确定最适合不同应用的系统，我们比较了经常使用的细胞类型中的Tet-on变体，这些细胞要么瞬时转染相关质粒，要么稳定地转导“一体式”慢病毒载体。

V10变异体在瞬时转染的细胞中表现最佳，在没有DOX的情况下没有背景活性，高DOX诱导活性和最高的折叠诱导活性。

由于V16系统对DOX的敏感度很高，如果细胞内DOX浓度很低，那么V16系统可能是首选。

V16在转导细胞中表现最好，在没有背景活性的情况下表现出最高的活性和对DOX的敏感性。

此外，在没有DOX的潜伏期后，V16表现出更强的基因表达诱导作用。

这项研究为选择适合不同细胞培养环境的最佳Tet-On系统提供了重要的发现。

对于大多数DNA存在于细胞中的应用来说，V10是最好的系统，而当Tet-on组分稳定整合到细胞基因组中时，V16可能是最佳的。

原文摘要：

The doxycycline (dox)-inducible Tet-On system is widely used to control gene expression in mammalian cells. This system is based on the bacterial Tet operon, which has been modified and improved for its function in eukaryotic cells. To identify the optimal system for different applications, we compared Tet-On variants in frequently used cell types that were either transiently transfected with the relevant plasmids or stably transduced with an "all-in-one" lentiviral vector. The V10 variant performed optimally in the transiently transfected cells and demonstrated no background activity without dox, high dox-induced activity and the highest fold-induction. Because of its very high dox-sensitivity, the V16 system may be preferred if only low intracellular dox concentrations can be reached. V16 performed optimally in the transduced cells and demonstrated the highest activity and dox-sensitivity without background activity. Moreover, V16 demonstrated more robust induction of gene expression after a latency period without dox. This study provides important findings for choosing the optimal Tet-On system for diverse cell culture settings. V10 is the best system for most applications in which the DNA is episomally present in cells, whereas V16 may be optimal when the Tet-On components are stably integrated in the cellular genome.

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参考文献：https://sci-hub.ren/10.1002/biot.201500236

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