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|Title: ||An Improved Antibody-based Method to Detect Whole Genome Cytosine Methylation in Mouse Embryonic Fibroblasts|
|Authors: ||CELIK, Selcen|
|Issue Date: ||17-Oct-2013|
|Publisher: ||School of Medicine|
Faculty of Medicine
|Abstract: ||Cytosine methylation (5meC) of the genome is an important epigenetic mark that acts as a regulator of gene expression and genome stability. The loss of methylation is reported to occur in the embryo following fertilisation. The immunological methods including antigenic retrieval with acid provide highly selective recognition of 5meC, and are commonly used to detect the changes in 5meC. However a recent study of the early embryo showed that a trypsin treatment after acid is required for increased antigenicity for 5meC epitope in mouse embryos.
This thesis presents the conditions required for reliable immunological analysis of global patterns of 5meC. It used a somatic cell model (mouse embryonic fibroblasts) and examined the effects of various cell growth states and the consequences of exposure of cells to various genotoxic stresses on staining of 5meC. This thesis showed that antigenic unmasking of 5meC and MBD1 (methyl-binding-protein1) with tryptic digestion revealed a heterogeneity in nuclear arrangements of methylation in somatic cells. These are accompanied by the changes in heterochromatin during DNA damage.
The results suggest a new model of DNA methylation within the mammalian genome. This represents multiple populations of genome which have different response to antigenic retrieval induced by some conditions, such cell growth and DNA damage.|
|Type of Work: ||PhD Doctorate|
|Type of Publication: ||Doctor of Philosophy Ph.D.|
|Appears in Collections:||Sydney Digital Theses (Open Access)|
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|CELIK Selcen - Final Thesis.pdf||8.46 MB||Adobe PDF|
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