Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters.
Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters.
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上下文相关的合成哺乳动物启动子的发现和验证。
DOI：
10.1101/2023.05.11.539703
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
English,JustinG
中科院分区：
文献类型：
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作者：
Zahm,AdamM;Owens,WilliamS;Himes,SamuelR;Rondem,KathleenE;Fallon,BradenS;Gormick,AlexaN;Bloom,JoshuaS;Kosuri,Sriram;Chan,Henry;English,JustinG
细胞转录使细胞能够适应各种刺激并维持稳态。转录因子与基因启动子中的转录应答元件（TREs）结合，启动转录。可以将源自天然TREs的合成启动子工程化以使用内源转录机制控制外源基因表达。该技术在生物学研究中广泛应用，包括报告基因测定，生物标志物开发和活细胞中的编程合成电路。然而，用于选择具有所需背景、幅度和刺激响应谱的最小尺寸合成启动子的可靠且精确的方法一直是难以捉摸的。在这项研究中，我们引入了一个大规模平行的报告基因检测库，包含6184个合成的启动子，每个小于250 bp的长度。该综合文库允许快速鉴定具有跨多个细胞系和刺激的最佳转录输出参数的启动子。我们展示了这个库的效用，以确定启动子激活独特的细胞类型，并响应代谢产物，促分裂原，细胞毒素，和激动剂的胺能和非胺能GPCR。我们进一步表明，这些启动子可以用于荧光素酶报告基因测定，引发50-100倍的动态范围响应于刺激。我们的平台是有效的，易于实施，并提供了一个解决方案，选择短长度的启动子与精确的性能，为众多的应用。
Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this library’s utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50–100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.