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Site-directed integration using the Cre/lox system in hematopoietic and embryonic stem cells Faulkes, Sharlene Marie

Abstract

Retroviral vectors have been most commonly used for performing gene function studies in hematopoietic cells and exploited for gene therapy of hematological inherited disorders. While unquestionably powerful, they suffer from several drawbacks including size limitations and the ability to confer long-term, predictable levels of transgene expression. The goal of this thesis was to develop an alternative gene transfer strategy that may overcome some of these hurdles and further provide an improved platform for performing gene function and gene regulation studies in hematopoietic cells. This strategy is based on the Cre/lox recombination system and can be summarized in two steps: (i) a simple retroviral vector is first used to introduce lox P target sites into the genome of target cells, (ii) followed by Cre mediated integration of exogenous DNA into the chromosomally placed lox P site. Initial studies demonstrated that site directed integration was feasible and applicable to many cell types, as it was shown to occur in both nonhematopoietic and hematopoietic cell lines, pluripotent embryonic stem (ES) cells, and in hematopoietic progenitors derived from the ES in vitro differentiation system. Moreover, this strategy is rapid, efficient and can be exploited to achieve predictable expression of a transgene upon re-targeting a locus. Gene regulatory mechanisms in hematopoietic cells were also studied using the ES in vitro differentiation model. A series of human p" globin expression vectors that are of interest for gene therapy of the hemoglobinopathies were assessed. The ES system was shown to be a potent model as the development of erythroid progenitors during differentiation correlated with the profile of globin gene expression and a large number of mature erythroid cells for RNA/protein analysis were readily generated. The Cre/lox system was shown to, be a more critical and informative method of evaluating constructs compared to random integration, since analysis was performed at the same integration site (assess transcription levels) and at more than one integration site (assess position effects). Moreover, Cre mediated integration was achieved with human P globin constructs that have been previously reported to be unstable in retroviral vectors and the size of which go beyond the current limitations for retroviral vectors.

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