While non-replicating extrachromosomal vectors remain in an extrachromosomal state they lead to a transient expression in proliferating cells. Retroviral and especially lentiviral vectors are the optimal tools to provide stable integration of expression cassettes into the host��s genome and thus guarantee the maintenance of the cassettes in replicating cells. Pseudotyping of lentiviral vectors allow to to transduce a broad range of different cell types from different species. The aim of this study was to provide and evaluate a strategy to efficiently implement a bimodal synthetic gene circuit in mammalian cells. For this purpose, three different methodologies were exploited. To precisely follow the induction/ repression properties of this gene circuit a destabilized enhanced green fluorescent protein mutant is coregulated, thereby reflecting the transcription of the transactivator. eGFP can be monitored in viable cells and facilitates the monitoring of expression on single cell level e.g. by flow cytometry. We implemented the autoregulatory expression module in NIH3T3 cells using lentiviral gene transfer. Cell clones were established and expression was monitored on the single cell level by flow cytometry. Increasing concentrations of the inducer were applied to investigate the pattern of expression response. Saturating inducer concentrations caused homogeneous activation and all cells showed eGFP expression. At non-saturating concentrations of the inducer the population was split and yielded a binary population distribution. By varying the inducer Pseudolaric-Acid-B concentration, the ratio of expressing to non-expressing cells could be adjusted. In particular, primary cells or even animals are so far not available for systems biology approaches. This is attributed not only to the genetic and phenotypic complexity of these natural systems but also to the lack of reliable methods to implement synthetic cassettes. Here, we show that expression cassettes conferring bimodal expression can be efficiently transduced with the help of lentiviral vectors. The results presented here were Oleuropein obtained with an immortalized cell line.