May Tobias, Eccleston Lee, Herrmann Sabrina, Hauser Hansjörg, Goncalves Jorge, Wirth Dagmar
Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
PLoS One. 2008 Jun 4;3(6):e2372. doi: 10.1371/journal.pone.0002372.
In order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties. To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression response which was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity. The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.
为了建立具有可预测特性的细胞和生物体，合成生物学利用可控的合成遗传装置。这些装置用于替代或干扰自然途径。或者，它们可以与内源性途径相互连接，以创建更高复杂性的人工网络。虽然这些方法在原核生物和低等真核生物中已经取得成功，但在哺乳动物系统甚至动物中实施此类合成盒仍然是一个主要障碍。这主要是由于缺乏可靠且高效地转导合成模块而不影响其调控特性的方法。为了为在哺乳动物系统中实施合成调控模块铺平道路，我们利用了合成模块的慢病毒转导。基于四环素调控系统的合成正反馈回路在慢病毒载体系统中得以实现，并稳定整合到哺乳动物细胞中。这个基因调控回路产生双峰表达响应。基于实验数据，开发了一个基于随机性的数学模型，该模型与实验结果相匹配并描述了实验结果。建模预测了滞后表达响应，并通过实验得到了验证。从而支持了该系统由随机性驱动的观点。这里展示的结果突出表明，三种独立工具/方法的结合有助于在哺乳动物细胞和生物体中可靠地安装具有可预测表达特征的合成基因回路。
