Title Page
Acknowledgement
CONTENTS
ABBREVIATIONS 9
Abstract 10
PART I : The analysis of X chromosome-inactivation related gene expression from early mouse single embryo by sex-determination 12
I. Introduction 12
II. Materials and Methods 14
II-1. Animals 14
II-2. Collection of single embryo 14
II-3. Total RNA isolation and RT-PCR 15
II-4. RFLP (Restriction fragment length polymorphism) 15
III. Results 16
III-1. Single-embryo analysis of the XCI genes from early mouse single embryo 16
III-2. Histone methylation and DNA methylation for X-chromosome inactivation 17
IV. Discussion 18
Figure legend 25
Reference 26
PART I. 국문초록 28
PART II : Estimating the total mouse DNA methylation according to the B1 repetitive elements 29
I. Introduction 29
II. Materials and Methods 31
II-1. Tissue and cell line 31
II-2. DNA Isolation and Bisulfite Modification 31
II-3. MSP 32
II-4. Pyrosequencing 32
III. Results and Discussion 34
III.1. The majority of B1 element for measuring DNA methylation in mouse total genome 34
III.2. Substantial method for measuring DNA methylation through the MSP and pyrosequencing with B1 element 35
III.3. MSP and pyrosequencing can detect DNA methylation decrease in cell line treated with 5-AzaC 36
Figure legend 42
Reference 44
PART II. 국문초록 47
Table 1. Semi-quantitative RT-PCR conditions for XCI-related gene expression 20
PART I 8
Figure1. RFLP analysis of RT-PCR products 21
Figure2. The expression pattern of the five XCI-related genes in every single 1-cell and 2-cell embryo 22
Figure3. The expression of Xist and Tsix genes in single 4-cell, 8-cell,and blastocyst of sex-determined embryos 23
Figure4. Gene expression pattern of SUV39H1, SET7, DNMT1, and ZFY in single 4-cell, 8-cell and blastocyst embryos 24
PART II 8
Figure1. The sequence of B1 element and primers for experiments 38
Figure2. The measurement of B1 DNA methylation in the mouse testis 39
Figure3. Quantitation of B1 DNA methylation changes by 5-AzaC treatment 40
Figure4. The relative B1 DNA methylation changes by5-AzaC treatment between the MSP and pyrosequencing data 41