A tunable dual-input system for 'on-demand' dynamic gene expression regulation
A tunable dual-input system for 'on-demand' dynamic gene expression regulation
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用于“按需”动态基因表达调节的可调双输入系统
DOI：
10.1101/404699
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Pedone E
中科院分区：
文献类型：
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作者：
Pedone E
细胞系统已经进化出许多适应环境刺激的机制，这些机制以基因表达的动态模式为基础。除了基因转录调控之外，蛋白质水平、动力学和定位的调节也是控制细胞功能的重要检查点。诱导型启动子的引入允许使用正交分子控制基因表达，促进其快速和可逆的操作以研究基因功能。然而，不同的蛋白质稳定性阻碍了体内观察到的蛋白质时间谱的生成。在这里，我们改进了 Tet-On 系统，在翻译后水平整合条件不稳定元件，并允许同时控制基因表达和蛋白质稳定性。我们证明，在哺乳动物细胞中，添加蛋白质稳定性控制可以实现更快的响应时间、完全可调和增强的动态范围，并改进基因表达的计算机反馈控制。最后，我们强调了双输入系统在调节小鼠胚胎干细胞信号通路成分水平方面的有效性。
Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.