Extracting input-output relationship of mammalian gene circuits using transient transfections

Input-output (I/O) relationship of natural and synthetic gene circuits is one of the key system characteristics. Data sets that allow determining these relationships are usually obtained by stably integrating genetic circuits into the cell genome. While stably integrated genetic circuits represent the “gold standard” for characterization, it is difficult and time consuming to establish stably integrated circuits; moreover such integration would normally be limited to immortalized cell lines due to the large number of passages required. Transient transfection, on the other hand, is a method that is time efficient and it could in principle be applied to cell lines and primary cells alike. However, it is not clear whether the extremely noisy data obtained in such experiments can be used to derive I/O relationships that would be comparable to the gold standard.

Here, we describe a data processing workflow that allows extracting I/O relationship from flow cytometry data obtained in transient transfections. We prove the workflow by computationally simulating both the “gold standard” and the transient transfection data using identical underlying mechanistic models of a number of representative gene circuits. The workflow is then applied to the in silico transient transfection data and the resulting I/O relationships are compared to the in silico gold standard data. We show that the results generated by our method closely overlap with the “gold standard” data, including in the case of multi-modal or bistable gene networks. Our workflow therefore enables determining I/O relationships of complex gene circuits using transient transfection only, greatly simplifying the design-characterization cycle.