Yang Zijie, Pan Yijie, Cai Yiming, Fu Tong, Feng Ao, Liu Yan, Wang Yiheng, Xiong Xinxuan, Cai Liang
School of Life Sciences, Fudan University, Shanghai 200438, China.
Sheng Wu Gong Cheng Xue Bao. 2018 Dec 25;34(12):1886-1894. doi: 10.13345/j.cjb.180290.
Transcriptional regulation is crucial for regulated gene expression. Due to the complexity, it has been difficult to engineer eukaryotic transcription factor (TF) and promoter pairs. The few availabilities of eukaryotic TF and promotor pairs limit their practical use for clinical or industrial applications. Here, we report a de novo construction of synthetic inhibitory transcription factor and promoter pairs for mammalian transcriptional regulation. The design of synthetic TF was based on the fusion of DNA binding domain and Kruppel associated box transcription regulating domain (KRAB). The synthetic promoter was constructed by inserting the corresponding TF response element after SV40 promoter. We constructed and tested five synthetic inhibitory transcription factor and promoter pairs in cultured mammalian cells. The inhibition capability and orthogonality were verified by flow cytometry. In summary, we demonstrate the feasibility of constructing mammalian inhibitory TF and promoter pairs, which could be standardized for advanced gene-circuit design and various applications in the mammalian synthetic biology.
转录调控对于基因表达的调控至关重要。由于其复杂性，构建真核转录因子（TF）和启动子对一直颇具难度。真核TF和启动子对的数量有限，限制了它们在临床或工业应用中的实际用途。在此，我们报告了用于哺乳动物转录调控的合成抑制性转录因子和启动子对的从头构建。合成TF的设计基于DNA结合结构域与Kruppel相关盒转录调节结构域（KRAB）的融合。合成启动子是通过在SV40启动子后插入相应的TF反应元件构建而成。我们在培养的哺乳动物细胞中构建并测试了五对合成抑制性转录因子和启动子对。通过流式细胞术验证了其抑制能力和正交性。总之，我们证明了构建哺乳动物抑制性TF和启动子对的可行性，这可为先进的基因电路设计及哺乳动物合成生物学中的各种应用实现标准化。
