Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.
Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.
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DOI：
10.1021/acssynbio.5b00266
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发表时间：
2016-05-20
影响因子：
4.7
通讯作者：
Chen YY
中科院分区：
文献类型：
--
作者：
Ede C;Chen X;Lin MY;Chen YY
诱导型转录系统在合成生物学回路中发挥着至关重要的作用。然而，大多数诱导型启动子是由有限的一组可靠的启动子部分构建的，其易受常见缺点如高基础表达水平（即，泄漏）。为了扩大调节哺乳动物基因表达的工具箱，并促进构建具有精确功能的哺乳动物遗传电路，我们定量表征了一组8个核心启动子，包括哺乳动物，病毒和合成来源的序列。我们证明，这种选择的核心启动子可以提供广泛的基础基因表达水平，并实现跨越两个数量级的倍数诱导梯度。此外，常用的部分如最小CMV和最小SV40启动子显示在诱导时实现稳健的基因表达，但也遭受高水平的泄漏。相比之下，合成启动子YB_TATA显示出在诱导状态下将低基础表达与高转录速率相结合，以实现与所有其他测试的启动子相比显著更高的倍数诱导比。当启动子与不同的遗传输出和不同的反应元件偶联时，以及在不同的宿主细胞类型和DNA拷贝数之间，这些行为保持一致。我们将这种对核心启动子特性的定量理解应用于对人类T细胞的成功工程化，所述人类T细胞通过特异性地在缺氧环境下的嵌合抗原受体信号传导对抗原刺激作出响应。在这项研究中提出的结果可以促进未来的哺乳动物合成生物学系统的设计和校准能够精确编程的功能。
Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning two orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.