Abstract
De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.
Highlights
De novo human artificial chromosomes (HAC) are small, extrachromosomal elements that contain a functional centromere, enabling their correct replication and segregation as stable normal chromosomes in human cells (Kouprina et al 2014; Grimes and Monaco 2005), together with the endogenous chromosomes
Autonomous HAC are characterised by high mitotic stability (Grimes and Monaco 2005; Moralli et al 2009), unlike other viral vectors which integrate into the host chromosome and may result in insertional mutagenesis (Baum 2007)
The herpes simplex virus type 1 (HSV-1) amplicon technology is a highly efficient delivery method based on transferring exogenous DNA packaged in HSV-1 amplicons into cells
Summary
De novo human artificial chromosomes (HAC) are small, extrachromosomal elements that contain a functional centromere, enabling their correct replication and segregation as stable normal chromosomes in human cells (Kouprina et al 2014; Grimes and Monaco 2005), together with the endogenous chromosomes. Autonomous HAC are characterised by high mitotic stability (Grimes and Monaco 2005; Moralli et al 2009), unlike other viral vectors which integrate into the host chromosome and may result in insertional mutagenesis (Baum 2007).
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