Title page
Acknowledgement
Contents
Abbreviations 11
Abstract 12
Ⅰ. Introduction 13
Ⅰ-1. Epigenetics and DNA methylation 13
Ⅰ-2. Global DNA methylation of rat genome by Identifier(ID) elements 14
Ⅰ-3. Epigenetic regulation during mouse oogenesis 15
Ⅱ. Materials and Methods 17
Ⅱ-1. Rat and pheochromocytoma (PC-12) cell line 17
Ⅱ-2. Genomic DNA isolation and bisulfite modification 17
Ⅱ-3. Unmodified and modified PCR amplification of the ID elements 18
Ⅱ-4. Direct sequencing 18
Ⅱ-5. Pyrosequencing 18
Ⅱ-6. Whole genome amplification (WGA) 19
Ⅱ-7. Mice and collection of oocytes 19
Ⅱ-8. mRNA isolation and one step RT-PCR 20
Ⅱ-9. Restriction fragment length polymorphism (RFLP) 20
Ⅲ. Results and Discussion 21
Ⅲ-1. Measuring the CpG methylation of ID elements 21
Ⅲ-2. Direct sequencing of bisulfite modified rat gDNA 21
Ⅲ-3. Measuring the CpG-3 methylation of the ID elements in rat by pyrosequencing 22
Ⅲ-4. The methylation change of ID elements by 5-Azacytidine treatment 24
Ⅲ-5. The expression pattern of three DNMT family in mouse oogenesis 24
Reference 36
Abstract in Korean 41
Table 1. Semi-quantitative RT-PCR conditions for three DNMT family expression 26
Figure 1. The experimental design for the methylation analysis of the ID elements. 27
Figure 2. Specific amplification of the ID elements with modified or unmodified rat gDNA. 28
Figure 3. Direct sequencing data of the ID element from bisulfite treated rat gDNA. 29
Figure 4. Methylation percentage of the CpG-3 site of the ID elements from six rat tissues (liver, lung, kidney, spleen, ovary and testis), blood, PC-12, and WGA gDNA. 30
Figure 5. The methylation percentage of CpG-3 site of the ID elements dependent on 5-AzaC treatment. 31
Figure 6. The expression pattern of the three DNMT family in GV and MⅡ stage oocytes. 32
Figure 7. RFLP analysis of RT-PCR products. 33