Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25430
標題: 小鼠胰臟核糖核酸水解酵素之表現以及應用核糖核酸干擾作用探討其對小鼠胚發育之影響
The expression of RNase 1 in mice and study of its effect on the development of mouse embryos by RNA interference
作者: 方榮達
Fang, Jung-Da
關鍵字: mouse embryo;鼠胚;ribonuclease;RNA interference;核糖核酸水解酵素;核糖核酸干擾作用
出版社: 畜產學系所
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J. Breukelman, M. P. Sasso, P. A. Jekel, E. Hodge, A. Furia and J. J. Beintema. 2001. Ruminant brain ribonucleases: expression and evolution. Biochim. Biophys. Acta. 1547: 95-103.
摘要: 
核糖核酸干擾作用(RNA interference, RNAi)為利用與內源性mRNA互補之雙股RNA(double stranded RNA, dsRNA)抑制特定基因表現之技術。而胰臟核糖核酸水解酵素(pancreatic ribonuclease, RNase 1)為RNase A家族之一員,可表現於9.5日齡之小鼠胚,但對其於早期胚發育之影響尚未瞭解。本研究除分析RNase 1於小鼠不同器官之表現外,擬藉由構築抑制RNase 1表現之載體,探討RNase 1對於早期鼠胚發育之影響。試驗一分別自八週齡ICR母鼠之不同組織器官抽取總RNA,經RT-PCR分析其RNase 1之表現,發現RNase 1於胰臟表現最多,其次依序分別為胃、卵巢、腎臟和心臟。試驗二於RNase 1 mRNA序列中挑選出6個適當之序列片段,分別構築於由U6啟動子(promoter)驅動之載體中,使其產製短的髮夾彎RNA(small hairpin RNA, shR-1~shR-6)。經轉染(transfection)至小鼠內皮胰臟細胞株(MS1 cell line)72 h後抽取其總RNA,以反轉錄聚合酶鏈鎖反應(reverse transcription polymerase chain reaction, RT-PCR)分析RNase 1之表現。結果顯示shR-1之構築最具抑制效果。試驗三利用顯微注射將shR-1中RNAi表現片段(U6-shR-1)導入小鼠原核期胚內,觀察其對胚後續發育能力之影響。經體外培養72 h,注射TE buffer或U6-shR-1片段之胚分裂率顯著低於未經任何處理之對照組(77與74% vs. 98%,P<0.05),而注射TE buffer與對照組之胚發育到桑椹期與囊胚期之百分比並無顯著差異(69% vs. 88%),但注射U6-shR-1者則顯著低於對照組(50% vs. 88%,P<0.05),顯示注射此外源性基因構築對早期鼠胚之發育有不良影響。將注射U6-shR-1之鼠胚移置到5隻代理孕母體內(N = 69),其中一隻於胚齡11.5天時犧牲,發現其子宮具有3個發育中止胚;其餘四隻代理孕母於懷孕期結束時,有兩隻分別產下兩隻仔鼠,但經PCR分析後,此四隻仔鼠之基因組並無U6-shR-1片段之嵌入。本研究結果顯示,小鼠RNase 1可表現於部份成體組織器官中,而本試驗所構築之dsRNA雖可有效抑制細胞株RNase 1之表現,但對於調控小鼠胚RNase1表現之效果與RNase1在早期胚發育所扮演之生理功能仍待進一步釐清。

RNA interference (RNAi), a sequence-specific gene silencing mechanism, has been developed to knock down cognate genes mediated by 21-23 nucleotide double-stranded RNAs (dsRNAs) homologous to target gene sequences. The pancreatic ribonulease, RNase 1, is a member of RNase A superfamily. It has been shown that RNase 1 expresses in mouse embryos at 9.5 d.p.c. and beyond, but little is known about its effects on the development of mouse embryos. Hence, the aim of this study was to investigate the expression profile of RNase 1 in the adult mouse tissues. Additionally, the physiological function of RNase 1 in the development of mouse embryos was examined by the constructed RNAi vectors for knocking down the expression of RNase 1. In Experiment 1, total RNAs from various organs of adult female ICR mice at 8 weeks of age were extracted and reverse transcription polymerase chain reaction (RT-PCR) was conducted to analyze the expression of RNase 1. The highest level of RNase 1 expression was in the pancreas, and in the stomach, ovary, kidney and the heart in sequence. In Experiment 2, six different 19-nucletide fragments of RNase 1 were chosen to construct RNAi vectors, from which the short hairpin RNAs (shRNAs) were produced by mouse U6 promoter. These vectors, designated as shR-1 to shR-6, were transfected into mouse pancreatic cell line, MS1, respectively. Total RNAs of transfected cells were extracted 72 h after transfection and the efficiency of RNAi from each construct was evaluated by RT-PCR. Results showed that shR-1 was the most effective construct in knocking down RNase 1 expression to 94% extent. In Experiment 3, the U6-shR-1 fragment digested from shR-1 vector was injected into the pronuclei of mouse zygotes. The cleavage rates in the U6-shR-1-injected and TE buffer-injected groups were significantly lower than that in the uninjected control group after 72 h culture in vitro (77 and 74% vs. 98%, P < 0.05). No significant differences of morula/blastocyst formation were found between TE buffer and control groups (69% vs. 88%, P > 0.05), but the development of morula/blastocyst in the U6-shR-1-injected group was significantly decreased, compared to the control group (50% vs. 88%, P < 0.05). The defective effect of U6-shR-1 on the development of preimplantation embryos was demonstrated. The U6-shR-1-injected embryos (N=137) developed to the morula and blastocyst stages (N=69) after 72 h culture in vitro were transferred into five pseudopregnant mice. One foster mouse was sacrificed at Day 11.5 of pregnancy, and three developed-arrested embryos were recovered. Four pups were born from two of the rest foster mothers. However, none of them carried the U6-shR-1 insert after PCR analysis. In this study, we found several other organs, including stomach, ovary, kidney and heart, also express pancreatic RNase 1 in mice. Six RNase 1-RNAi expression vectors with various inhibitory efficiency in MS1 cells were constructed. Introduction of shRNA targeting RNase 1 into mouse zygotes might be defective to the development of preimplantation embryos. The influence of RNase 1 on the postimplantation development requires further investigation.
URI: http://hdl.handle.net/11455/25430
其他識別: U0005-2807200612101900
Appears in Collections:動物科學系

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