Bimodal and hysteretic expression in mammalian cells from a synthetic gene circuit

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 responsewhich 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.