(87e) Properties of Intragenic microRNA-Based Gene Regulation in Single Cells and Population Measurements

Intragenic microRNAs can regulate their host genes through targets in the gene's 3â?? untranslated region (UTR). This regulation is an incoherent feed-forward loop (IFFL), with a transcription factor producing both the host gene and the negative regulator of the output, the microRNA. To characterize the properties of a microRNA based IFFL, we used CRISPR/ Cas9 to stably integrate an engineered genetic circuit into the AAVS locus of human kidney cells. The genetic circuit consists of two fluorescent proteins produced by a bi-directional TRE promoter in response to the transcription factor rtTA and the small molecule doxycycline. One protein, DsRed, is produced as two exons with an intronic synthetic microRNA; the other protein, AmCyan, is used as a control. The DsRed gene has microRNA targets in its 3â??UTR. We performed flow cytometry measurements on our circuit and control monoclonal cell lines under a range of doxycycline concentrations and at multiple time points. We also performed time-lapse fluorescence microscopy measurements on our cell lines under full doxycycline induction. We analyzed the data to study the properties of the intragenic microRNA based regulation, in particular how the IFFL regulation affects the transient behavior of the output in single cells, the type of distribution of the output in the population, and finally the impact on the noise levels.