Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland.
Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH, 4058, Basel, Switzerland.
Metab Eng. 2021 May;65:99-110. doi: 10.1016/j.ymben.2021.03.009. Epub 2021 Mar 17.
Advances in synthetic biology have enabled robust control of cell behavior by using tunable genetic circuits to regulate gene expression in a ligand-dependent manner. Such circuits can be used to direct the differentiation of pluripotent stem cells (PSCs) towards desired cell types, but rational design of synthetic gene circuits in PSCs is challenging due to the variable intracellular environment. Here, we provide a framework for implementing synthetic gene switches in PSCs based on combinations of tunable transcriptional, structural, and posttranslational elements that can be engineered as required, using the vanillic acid-controlled transcriptional activator (VanA) as a model system. We further show that the VanA system can be multiplexed with the well-established reverse tetracycline-controlled transcriptional activator (rtTA) system to enable independent control of the expression of different transcription factors in human induced PSCs in order to enhance lineage specification towards early pancreatic progenitors. This work represents a first step towards standardizing the design and construction of synthetic gene switches for building robust gene-regulatory networks to guide stem cell differentiation towards a desired cell fate.
合成生物学的进展使得通过使用可调谐的遗传电路以配体依赖的方式调节基因表达来实现对细胞行为的稳健控制成为可能。这样的电路可用于指导多能干细胞（PSCs）向所需的细胞类型分化，但由于细胞内环境的可变性，在 PSCs 中合理设计合成基因电路具有挑战性。在这里，我们提供了一个基于可调转录、结构和翻译后元件组合的在 PSCs 中实现合成基因开关的框架，这些元件可以根据需要进行工程设计，我们使用香草酸控制转录激活剂（VanA）作为模型系统。我们还表明，VanA 系统可以与成熟的反四环素控制转录激活剂（rtTA）系统多路复用，以实现对人诱导 PSCs 中不同转录因子表达的独立控制，从而增强向早期胰腺祖细胞的谱系特化。这项工作代表着朝着标准化设计和构建合成基因开关以构建稳健的基因调控网络以指导干细胞向所需细胞命运分化迈出的第一步。
