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Approaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behaviour. The majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow, and easy to transfect. Additionally, genetic circuits that integrate into the genome of these immortalized lines remain functional for many generations, often for the lifetime of the cells. However, when genetic circuits are integrated into the genome of stem cells, gene silencing is observed within a few generations. To investigate the stability of genetic circuits and their propensity to silence in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to stabilize the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome silencing of genetic circuits in pluripotent stem cells that enable the possibility of leveraging synthetic biology in pluripotent stem cells to meet therapeutic needs.

Approaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behaviour, however, the majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow and easy to transfect. Genetic circuits that integrate into the genome of these immortalized cell lines remain functional for many generations, often for the lifetime of the cells, yet when genetic circuits are integrated into the genome of stem cells gene silencing is observed within a few generations. To investigate the reactivation of silenced genetic circuits in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to reactivate the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome the silencing of genetic circuits in pluripotent stem cells that may enable the use of genetic circuits in pluripotent stem cells for long-term function.

No abstract