TITLE PAGE
ACKNOWLEDGEMENT
CONTENTS
ABSTRACT 9
I. INTRODUCTION 11
II. MATERIALS AND METHODS 14
II.1. Animals and total RNA isolation 14
II.2. RT-PCR analysis 14
II.3. Tissue preparation and in situ hybridization 17
II.4. Immunohistochemistry 18
II.5. Preparation of FAM-labeled siRNA 18
II.6. Cell culture and siRNA transfection 20
II.7. Statistical analysis 21
III. RESULTS 22
III.1. RT-PCR analysis for genes related to the cell size growth and CCN family 22
III.2. In situ hybridization 24
III.2.1 Genes related to cell size growth 24
III.2.2 Genes in CCN family 27
III.3. Protein expression of Cyr61 and Wisp1 29
III.4. Preparation of FAM-labeled cyr61 siRNA 31
III.5. RNAi for endogenous cyr61 in NIH 3T3 cells 33
IV. DISCUSSION 36
V. CONCLUSION 40
REFERENCE 41
SUMMARY IN KOREAN 46
Table 1. Primer sequences and RT-PCR conditions 16
Figure1. Heat map of genes related to the cel lsize growth 13
Figure2. Differential expression pattern according to ovarian developmental stages by semiquantitative RT-PCR 23
Figure3. In situ hybridization of genes related to cell size growth in the day 5 ovaries 25
Figure4. In situ hybridization of genes related to cell size growth in the day14 ovaries 26
Figure5. In situ hybridization of genes for CCN family members in the day 14 ovaries 28
Figure6. Immunohistochemistry for Cyr61and Wisp1 in the day5 ovary 30
Figure7. Generation of cyr61 siRNA 32
Figure8. Effect of RNAi for cyr61 on the expression of cyr61, ctgf, and wisp 1 mRNA in NIH 3T3 cells 34
Figure9. Microphotographs of the NIH 3T3 cells after RNAi for cyr61 35