Gene Silencing of Cyclooxygenase-2 mRNA by RNA Interference in Bovine Cumulus-Granulosa Cells
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概要
- 論文の詳細を見る
Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F2α (PGF 2α) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF2 α concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF2α concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF2α concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.
- 日本繁殖生物学会の論文
- 2007-12-01
著者
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OKUDA Kiyoshi
Laboratory of Reproductive Endocrinology, Okayama University
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高橋 昌志
(財)日本自動車研究所
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SAKATANI Miki
National Agricultural Research Center for Kyushu Okinawa Region
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TAKAHASHI Masashi
National Agricultural Research Center for Kyushu Okinawa Region
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Okuda Kiyoshi
Laboratory Of Reproductive Endocrinology Graduate School Of Natural Science And Technology Okayama U
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Sakatani Miki
National Agricultural Res. Center For Kyushu Okinawa Region
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Kobayashi Shuji
Department of Asian Studies, Chukyo Women's University
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Kobayashi Shu-ichi
National Livestock Breeding Center
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Okuda Kiyoshi
Laboratory Of Reproductive Endocrinology Graduate School Of Natural Sciences And Technology Okayama
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KOBAYASHI Shu-ichi
Research Team for Effects of Climate Change on Agriculture, National Agricultural Research Center fo
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SAKATANI Miki
Research Team for Effects of Climate Change on Agriculture, National Agricultural Research Center fo
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TAKAHASHI Masashi
Research Team for Effects of Climate Change on Agriculture, National Agricultural Research Center fo
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高橋 昌志
九州沖縄農業研究センター 畜産飼料作研究部 繁殖技術研究室
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Takahashi Masashi
Dep. Of Animal And Grassland Res. National Agricultural Center For Kyushu Okinawa Region
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高橋 昌志
(独)農業・食品産業技術総合研究機構九州沖縄農業研究センター 暖地温暖化研究チーム
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