caa a22 4500 606190279 CHVBK 20210128100854.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s10577-014-9456-2 doi (NATIONALLICENCE)springer-10.1007/s10577-014-9456-2 Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology [Elektronische Daten] [Daniela Moralli, Zoia Monaco] 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. The Author(s), 2015 gene therapy nationallicence gene expression nationallicence viral replication nationallicence viral vector nationallicence AAV : Adeno-associated virus nationallicence FISH : Fluorescence in situ hybridization nationallicence HAC : Human artificial chromosome nationallicence hESc : Human embryonic stem cells nationallicence HSV-1 : Herpes simplex virus type 1 nationallicence HPRT : Hypoxanthine phosphoribosyltransferase nationallicence Moralli Daniela The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, OX3 7BN, Oxford, UK aut Monaco Zoia Department of Biomedical Engineering, Tufts University, 4 Colby Street, 02155-6013, Medford, MA, USA aut Chromosome Research Springer Netherlands 23/1(2015-02-01), 105-110 0967-3849 23:1<105 2015 23 10577 https://doi.org/10.1007/s10577-014-9456-2 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10577-014-9456-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Moralli Daniela The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, OX3 7BN, Oxford, UK aut NATIONALLICENCE 700 1- Monaco Zoia Department of Biomedical Engineering, Tufts University, 4 Colby Street, 02155-6013, Medford, MA, USA aut NATIONALLICENCE 773 0- Chromosome Research Springer Netherlands 23/1(2015-02-01), 105-110 0967-3849 23:1<105 2015 23 10577