CRISPR/Cas9 and human induced Pluripotent Stem Cells Sample Clauses

CRISPR/Cas9 and human induced Pluripotent Stem Cells. The CRISPR/Cas9 gene editing system consists of two components; a Cas9 protein that is guided by a guide RNA to a piece of DNA of interest where it will introduce a double stranded break. Introduction of these breaks will activate either non-homologous end joining or the homologous recombination pathway. Non-homologous end joining is prone to errors as no template is utilized. Often point mutations, deletion or insertions are left at the site that is targeted by the Cas9 protein (17). When homologous recombination is utilized by the cell, a template is used to repair the break. This template can be provided to insert a custom sequence within the break. Because chromosomal translocations are a result of double stranded breaks and non-homologous end joining, CRISPR/Cas9 can be used to introduce chromosomal translocations with reasonable efficiency (figure 1.3) (18). Introducing chromosomal translocations to model gene fusions has large advantages over using an expression system because expression and regulation of the fusion gene remains under control of the original promoter and therefore represents expression and regulation as found in tumor cells. CRISPR/Cas9 has been used to model fusion driven tumors previously. In mesenchy- Figure 1.3: Induction of translocations using CRISPR/Cas9. Two break points are intro- duced and both the NHEJ and HR can lead to the formation of a translocation. mal stem cells CRISPR/Cas9 was used to introduce the EWSR1-WT1 fusion, to model desmoplastic small round cell tumors (19). The EWSR1-FLI1 fusion was introduced in human mesenchymal stem cells to model Xxxxx sarcoma (20). However, because Xxxxx sarcoma does not show evident differentiation towards a normal cell type it is not possible to study the effects of the fusion gene on cells with matching differentiation to Xxxxx sar- coma cells, which influences the observed effects of the fusion gene. In chapter six of this thesis the SERPINE1-FOSB fusion is introduced in hiPSCs to overcome the limitations of using HUVECs combined with a lentivirus delivery system as a model to study pseudomyo- genic hemangioendothelioma. Because pseudomyogenic hemangioendothelioma shows en- dothelial differentiation the functional effects of the SERPINE1-FOSB fusion were studied in human induced pluripotent stem cells differentiated to endothelial cells. As indicated before, one of the limitations of using HUVECs is their limited life- span. To overcome this issue human induced pluripotent stem ...
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