An engineered epigenetic transgene switch in mammalian cells

Abstract

In multicellular systems cell identity is imprinted by epigenetic regulation circuits, which determine the global transcriptome of adult cells in a cell phenotype-specific manner. By combining two repressors, which control each other's expression, we have developed a mammalian epigenetic circuitry able to switch between two stable transgene expression states after transient administration of two alternate drugs. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch-specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models. Epigenetic transgene control through toggle switches is an important tool for engineering artificial gene networks in mammalian cells.

A bistable genetic switch based on designable DNA-binding domains

“…This type of design for a genetic toggle switch has been implemented in bacteria 8 as well as in mammalian cells 9 . Although several natural prokaryotic transcription factors have been used to assemble genetic circuits [8][9][10][11] , the number of orthogonal transcription factors for the toolbox should be The mutual repressor switch topology, consisting of two TALE repressor genes placed downstream of the constitutive CMV promoter with an operator for the opposing TALE repressor upstream of the promoter, and (b) TALE-based toggle with additional constructs for the external control and two different fluorescent protein reporters. The overlay of the microscopy images of the TALE-based mutual repressor switch indicates the simultaneous expression of both fluorescent reporter proteins in individual cells in the absence of external regulation.…”

A synthetic mammalian gene circuit reveals antituberculosis compounds

“…Synthetic gene circuits have dramatically increased progress on gene-function relationships in the postgenomic era (8)(9)(10)(11)(21)(22)(23)(24)(25). They also continue to provide the key parts for synthetic biologists to decipher natural gene network dynamics (8)(9)(10) and to reprogram cellular function for production of important precursor drugs (23).…”

“…They also continue to provide the key parts for synthetic biologists to decipher natural gene network dynamics (8)(9)(10) and to reprogram cellular function for production of important precursor drugs (23). We have engineered a synthetic gene network in human cells for screening of functional antimicrobial agents with precise target specificity, undetectable cytotoxicity, and the capacity to reach the cytosol to eliminate intracellular pathogens.…”

Systems and Synthetic biology: tackling genetic networks and complex diseases

“…In 2004, Kramer et al (Kramer et al, 2004), building on their previous work carried out on the construction of novel inducible systems, constructed the first switch in mammalian cells, which has the same design as the one previously described and implemented by Gardner et al (2000) in E. coli. The switch uses as promoters the streptogramin PpirON and the macrolide PetrON-inducible promoters, which direct expression of two repressors: E-Krab and PIP-Krab.…”