Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems.
A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism.
B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/16785 |
| Date | 20 January 2009 |
| Creators | George, Sophia |
| Contributors | Nagy, Andras |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 18996438 bytes, application/pdf |
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