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This dissertation, Establishment of Target DNA Methylation of PDX1 on Human Embryonic Stem Cells by Chor-ying, Crystal, Wong, 黃楚瑩, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: DNA methylation, one of the epigenetic modifications, has been linked to regulate gene expression which controls the development of lineage-specific cells as well as disease. The theory of developmental origins of health and disease suggests that in utero hyperglycemic conditions can induce epigenetic changes which lead to the risk of developing adult onset diseases later in life. Type 2 Diabetes (T2D) has been associated with the impaired development of pancreatic β-cells and hypermethylation of the pancreatic-duodenum homeobox 1 (PDX1). However, the causative effect of DNA methylation on the development of T2D is elusive. The aim of this study was to establish the targeted methylation of PDX1 in human embryonic stem cells (hESC) in hopes of using the established method to further understand the development of the disease. The hESC line (VAL3) used in this study was first characterized. The non-differentiated VAL3 expressed well-known pluripotent markers, OCT4 and NANOG. Upon definitive endoderm (DE) differentiation, the first crucial step for generating gastrointestinal tracts including the pancreas, significant induction of the DE markers (SOX17, FOXA2, MIXL1 and CXCR4) was found. Further pancreatic progenitor (PP) differentiation using small molecule ILV also induced the PP marker, PDX1 expression. The DNA methylation status of PDX1 was also analyzed in non-differentiated VAL3 and differentiating DE and PP cells using bisulfite conversion followed by DNA sequencing. The results showed that the CpG-rich proximal promoter region of the PDX1 were hypomethylated at all three developmental stages. The role of DNA methylation was further demonstrated by the treatment of DNA methyltransferase inhibitor 5- azacytidine during DE and PP differentiation. It was found that 5-azacytidine had no effect on the expressions of the pluripotent, DE and PP markers. The CpG-rich proximal promoter region of the PDX1 was then selected for the establishment of target methylation method in VAL3. Restriction enzyme digestion suggested that majority of the CpG-rich proximal promoter region of the PDX1 was methylated after in vitro methylation. The unmethylated and methylated PDX1 promoter DNA fragments were then fluorescently labeled with Cy5 and transfected into non-differentiated VAL3 cells. Fluorescence Activated Cell Sorting (FACS) results showed that 7.6% and 3.3% of Cy5 positive cells were obtained from unmethylated and methylated DNA transfected VAL3, respectively. The Cy5 positive cells were reseeded and colonies from single cells were formed. The Cy5 signals were only detected 24h after reseeding but not afterwards. The colonies were growing in the laboratory and awaiting the confirmation of PDX1 DNA methylation by bisulfide sequencing. In conclusion, this study showed that CpG rich PDX1 proximal promoters are hypomethylated in non-differentiated VAL3, differentiated DE and PP cells. An in vitro target methylation of PDX1 promoter was established. The transfection of the methylated DNA fragment did not affect the viability of VAL3. The results obtained in the current study formed the basis for the functional study of pre-deposited DNA methylation in the differentiation potential of hESC. Subjects: Embryonic stem cellsEmbryonic stem cellsDNA - Methylation

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