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Title: Characterisation of transgene expression in human embryonic stem cells by combining gene targeting and site-specific recombination
Author: Di Domenico, Alexandra Isabelle
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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Predictable levels of transgene expression will be essential for full exploitation of human ES cells (hES) in basic research and medicine. In practice however, transgene expression in mammalian cells is often found to be silenced, low or variegated presenting a serious drawback in this area of research. Unpredictability of transgene expression arises mostly as a consequence of variable transgene copy number and/or inhibitory effects of surrounding chromatin structures in which the transgene has randomly integrated. We have addressed this issue in hES cells, by combining gene targeting and site-specific recombination at the (3-casein and the hypoxanthine phosphoribosyltransferase {hprt) genes. A neomycin expression cassette flanked by heterospecific lox sites is first introduced by homologous recombination (HR). In a subsequent step, recombinase-mediated cassette exchange (RMCE), permits the exchange of the neomycin gene with a transgene containing a blasticidin-oct-4-GFP expression cassette flanked by the same set of heterospecific lox sites. Using a promoter trap strategy, site-specific integrants can be directly selected with blasticidin. While we failed to target the p-casein gene in hES cells, we obtained homologous-recombinants with targeting frequencies of up to 0.4 % at the hprt gene in the male hES cell line (HI). F/prMargeted HI cells were shown to maintain a normal karyotype; to exhibit common hES cell surface markers; and to differentiate down the osteogenic lineage pathway. Additionally, subsequent targeted correction of hprt-targeted HI mutant cells was achieved, restoring structure and function of the hprt gene. Following RMCE, site-specific integrant were recovered with 100 % efficiency using a promoter trap strategy at the hprt locus. However, site-specific integrants were characterised by a progressive down-regulation of GFP, possibly as a result of transcriptional interference from the BSD cassette or inhibitory elements present at the site of integration. Further work will be required to investigate the nature of this silencing. Targeting transgenes with reliable transgene expression at specific loci in human ES cells will be important for modifying specific ES cell-derived tissues for therapeutic applications in regenerative medicine. Engineering the hprt locus should prove very useful for addressing issues such as directed differentiation, immunotolerance as well as protection against tumorigenesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available