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UBC Theses and Dissertations

Development of tools in mouse for future gene therapy : promoters for the CNS, and novel expression models of neural stem cell regulator, NR2E1. de Leeuw, Charles Norval


Three challenges exist for human neurobiology, specifically in the areas of genomics and genetic medicine: understanding of genome regulation, understanding of central nervous system (CNS) development, and the lack of promoters for human gene therapy. To address these issues, we used computational biology strategies, primarily involving phylogenetic footprinting, to identify putative regulatory elements in genes with a regionalized or cell-type specific expression pattern. We generated human MiniPromoter constructs, less than 4 kilobases in size, and made genetically engineered mice by single-copy knock-in at the mouse Hprt locus. Neuroanatomical analyses were performed on brain and eye, primarily. Using this strategy, we generated 50 novel MiniPromoters for use in driving gene therapy constructs. Lastly, we demonstrated retained specificity of three retinal ganglion cell layer MiniPromoters when these were moved from knock-ins to an adeno-associated viral vector, exemplifying the utility of these constructs in other systems. In order to study CNS development, we chose to functionally analyze non-endogenous expression of the neural stem cell regulator NR2E1 in mice. We employed a DCX-based MiniPromoter from the Pleiades Promoter Project, in addition to the ubiquitous CAG promoter, to drive ectopic and ubiquitous expression of human NR2E1. DCX-based expression of human NR2E1 did not result in any overt phenotypes and was unable to rescue the brain and eye defects observed in Nr2e1frc/frc mice. In contrast, the CAG promoter resulted in embryonic death at ~E8, with failure of neural tube closure. We showed that expression of NR2E1 has negative effects on embryonic stem cell growth. Furthermore, we observed altered Pax6 expression in NSCs and embryos. Future work on promoter design and NR2E1 biology will advance our knowledge of genome regulation and CNS development.

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