Title Page
Contents
Abstract 9
Ⅰ. INTRODUCTION 11
Ⅱ. MATERIALS AND METHODS 13
1. Animal 13
2. Oocyte Collection and Culture 13
3. RNA interference 14
4. Microinjection of siRNA and in vitro culture 14
5. RNA extraction, reverse transcription and quantitative real time polymerase chain reaction 15
6. Epifluorescence Microscopy of Oocyte 17
7. Immunofluorescence staining 17
8. Statistical analysis 18
Ⅲ. RESULTS 19
1. Expression of 1 Trim71 mRNA in mouse tissues. 19
2. Expression of Trim71 mRNA in mouse oocytes and pre-implantation embryos. 21
3. Expression of Trim71 mRNA during oocyte growth. 23
4. The effect of Trim71 knock-down during in vitro maturation. 25
Ⅳ. DISCUSSION 33
Ⅴ. CONCLUSION 35
REFERENCES 36
ABSTRACT IN KOREAN 38
Table 1. Primer sequences of genes used for RT-PCR and qRT-PCR 16
Table 2. In vitro maturation of preincubated oocyte oocyte in 5µM milrinone for 8, 16 and 24 hours. 31
Table 3. In vitro maturation of mouse oocytes after injection of Trim71 siRNA in GV oocytes. 32
Fig. 1. Trim71 expression in mouse tissues. 20
Fig. 2. Differential expression of Trim71 during oocyte maturaiton and pre-implantation embryo development. 22
Fig. 3. Trim71 expression during oocyte growth. 24
Fig. 4. Oocyte maturation of preincubated oocyte in 5µM milrinone for 8, 16 and 24 hours. 27
Fig. 5. Trim71 RNAi in GV oocytes. 28
Fig. 6. Effect of Trim71 knockdown on oocyte maturation. 29
Fig. 7. Immunofluorescence image of Trim71 siRNA knockdown effect in MII oocyte. 30