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Title: Analysis of artificial chromosomes in human embryonic stem cells
Author: Mandegar, Mohammad Ali
ISNI:       0000 0004 2722 0601
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.
Supervisor: Larin Monaco, Zoia Sponsor: Natural Sciences and Engineering Council of Canada (NSERC) ; EPA Cephalosporin Award
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cell Biology (see also Plant sciences) ; Genetics (life sciences) ; Mammalian chromosome ; Human embryonic stem cells ; Human artificial chromosomes (HAC) ; Herpes simplex virus type 1 (HSV-1) amplicon ; Genetic manipulation ; Gene therapy ; Chromosomal analysis