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

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.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation

SummaryPax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7−/− satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle