Abstract
The enabling technologies of synthetic biology are opening up new opportunities for engineering and enhancement of mammalian cells. This will stimulate diverse applications in many life science sectors such as regenerative medicine, development of biosensing cell lines, therapeutic protein production, and generation of new synthetic genetic regulatory circuits. Harnessing the full potential of these new engineering-based approaches requires the design and assembly of large DNA constructs-potentially up to chromosome scale-and the effective delivery of these large DNA payloads to the host cell. Random integration of large transgenes, encoding therapeutic proteins or genetic circuits into host chromosomes, has several drawbacks such as risks of insertional mutagenesis, lack of control over transgene copy-number and position-specific effects; these can compromise the intended functioning of genetic circuits. The development of a system orthogonal to the endogenous genome is therefore beneficial. Mammalian artificial chromosomes (MACs) are functional, add-on chromosomal elements, which behave as normal chromosomes-being replicating and portioned to daughter cells at each cell division. They are deployed as useful gene expression vectors as they remain independent from the host genome. MACs are maintained as a single-copy and can accommodate multiple gene expression cassettes of, in theory, unlimited DNA size (MACs up to 10 megabases have been constructed). MACs therefore enabled control over ectopic gene expression and represent an excellent platform to rapidly prototype and characterize novel synthetic gene circuits without recourse to engineering the host genome. This review describes the obstacles synthetic biologists face when working with mammalian systems and how the development of improved MACs can overcome these-particularly given the spectacular advances in DNA synthesis and assembly that are fuelling this research area.
Highlights
Synthetic biology is an emerging area of research whose main aim is the design, construction and characterization of synthetic genetic circuits that once integrated into the cellular host produce desirable outputs in a robust and predictable manner.[1−3] In recent years, synthetic biology applied to mammalian cells has rapidly evolved and achieved impressive advancements and proof-of-principle studies for the generation of complex and diverse genetic devices.[1−3] Several functional genetic modules such as toggle switches,[4] boolean logic gates,[5] hysteretic switches,[6] oscillators[7] and light induced optogenetic switches[8] have been successfully developed in mammalian cells
We will first discuss the issues of scalability, orthogonality and predictability that must be addressed for the implementation of more complex synthetic genetic circuits in mammalian systems
Genetic engineering has traditionally been limited to small-scale locus-specific or random changes aimed in a gene-centric manner, rather than enhancement or delivery of novel synthetic genetic circuits with multiple parts and control elements
Summary
Review reproducible and cheaper ways to engineer living materials.[9]. Synthetic biology has been regarded as a genetic engineering in overdrive enabling, through advances in de novo DNA synthesis and DNA assembly,[10,11] the generation of novel proteins, regulatory elements and genetic circuits, repurposed for specific goals that are designed and engineered without being constrained by overwhelming complexity of natural cellular physiology. After being transfected with 30−200 Kb alpha-DNA, by mechanisms not yet fully understood, the cell recognizes the transgenic DNA as centromeric seeding the deposition of specific protein and epigenetic markers on the exogenous DNA These modifications will lead to the generation of a fully active centromere, converting the vector into an artificial chromosome.[78] The resulting HAC will be circular if the input DNA is cloned into a BAC or linear when a YAC carrying telomeric sequences is used as a vector to introduce the alphoid DNA in the target cells[79−82] (Figure 2). All these challenges call for interdisciplinary teams to work together to address, and together to push this field forward
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