Figure 4.

Transcriptional regulation and genomic rearrangement for a 7-gene circuit integrated into the landing pad of HEK293FT chassis cell line. (A) Schematic representation of the integrated circuit. In its initial state, the circuit expresses puromycin resistance gene, Cerulean, rtTA3 and Blasticidin resistance gene. After dox induction, rtTA3 activates TREt promoter and triggers expression of EYFP and B3 recombinase (Step 1). This leads to the intermediate state, during which B3 integrase mediates excision of the Cerulean cassette by recombination of the two attB3 sites. Subsequently, mKate2 gene is expressed. When dox is removed, EYFP is no longer expressed. In the final state, mKate2 is the only reporter that is expressed. (B) Fluorescent microscopy images and flow cytometry results confirmed the correct behavior of the circuit and show homogeneity of transgene expression in the polyclonal cell population. (Intermediate state: 3 days after dox induction; Final state: 7 days after dox removal.)