Engineered Promoters for Potent Transient Overexpression

The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods.核心启动子（core promoter）通常被定义为招募RNA聚合酶II（RNA Polymerase II）以启动转录的区域，其在基因表达调控中发挥关键作用。核心启动子由多种短DNA序列的不同组合构成，这些序列被称为核心启动子元件（core promoter elements）或基序（motifs），可为每个启动子赋予特定的功能特性。此前有研究围绕通过核心启动子调控基因表达水平的能力展开探索，由此设计出了将多种核心元件整合为单一核心启动子的强效合成型核心启动子。本研究设计了一款新型核心启动子，命名为超级核心启动子3（super core promoter 3, SCP3），它将四种核心启动子元件（TATA框（TATA box）、Inr、MTE以及DPE）整合为单一启动子，可驱动持久且强效的基因表达。我们通过构建由不同核心启动子驱动的增强型绿色荧光蛋白（EGFP）表达载体，分析了核心启动子结构对报告基因表达时序动态的影响。我们在不同人类细胞系中采用活细胞成像与流式细胞术分析，证实了超级核心启动子（SCPs），尤其是新型SCP3，可驱动异常强劲的长期EGFP表达。值得注意的是，本研究首次在瞬时转染的哺乳动物细胞中验证了长期基因表达，这表明经工程改造的核心启动子可为需要长时间强效表达的生物技术及基因治疗相关应用提供一种新型非病毒策略。