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標題: 應用核糖核酸干擾作用抑制小鼠胚血管生成素之表現
Inhibition of angiogenin gene expression of mouse embryos by RNA interference
作者: 王世寬
Wang, Shih-Kuan
關鍵字: Angiogenin;血管生成素;RNA interference;RNase A gene family;mouse embryo;siRNA;核糖核酸干擾作用;RNase A基因家族;鼠胚;小分子干擾性核糖核酸
出版社: 畜產學系
核糖核酸干擾作用(RNA interference, RNAi)為一快速抑制基因表現技術,目前被廣泛應用於基因功能之研究與基因治療。小鼠血管生成素(angiogenin, Ang)為常存於血液循環之蛋白質。Ang、RNase 1與RNase 4同屬於RNase A gene family,且同位於小鼠第14對染色體上,其主要生理活性為促血管生成與水解核糖核酸。試驗壹利用RT-PCR分析Ang、RNase 1與RNase 4於小鼠著床前與著床後胚胎之表現。結果顯示Ang可持續表現於著床前(E1.5、E2.5與E3.5)與著床後胚胎(E7.5、E9.5與E12.5),而RNase 4則於E7.5天開始微量表現,RNase 1則可表現於E9.5與E12.5天之胚胎中。進一步分析3種小鼠細胞株,於CT-26與3T3細胞株均可偵測到Ang、RNase 1與RNase 4之表現,於B16F10細胞株則可偵測到RNase 1與Ang之表現,但無RNase 4之表現。轉染3種Ang-RNAi表現載體(pUC-ANG1, -ANG2, -ANG3)於小鼠CT-26與B16F10細胞株中並經24 h培養後,發現此3種RNAi表現載體均可有效(P<0.05)抑制此2種細胞株Ang之表現,但對不同細胞株之抑制效率不同。經轉染不同Ang-RNAi表現載體後,CT-26 Ang之表現由低至高分別為22%(pUC-ANG1)、47%(pUC-ANG2)與47%(pUC-ANG3),而於B16F10則為18.07%(pUC-ANG2)、33%(pUC-ANG3)與57%(pUC-ANG1)。於B16F10中pUC-ANG2與pUC-ANG3對Ang表現之抑制效果顯著高於pUC-ANG1。試驗貳將U6-ANG1 RNAi表現卡匣(expression cassette)以顯微注射法導入原核期鼠胚中,探討Ang對鼠胚發育之影響。經注射之鼠胚於體外培養72 h後,單獨注射U6-ANG1基因或U6-ANG1與EGFP基因共注射組(co-injection)之囊胚形成率與對照組者無顯著差異,但共注射組鼠胚之分裂率顯著低於對照組者(67% vs. 83%, P<0.05),而發育至桑椹胚之百分率亦以處理組(單一基因注射組vs. 共注射組, 65 vs. 58%)顯著低於對照組(73%),且兩個基因注射組間亦有顯著差異(P<0.05),顯示注射外源性基因對早期鼠胚之體外發育有不良之影響。共注射處理後之鼠胚經胚移置後,產下之仔鼠以PCR分析,其中四隻仔鼠(4/24)為具有EGFP基因嵌合之轉基因鼠,但未見U6-ANG1 RNAi表現卡匣嵌合之訊號。本研究結果顯示,著床前與著床後之小鼠胚胎均可偵測出Ang之表現。雖然應用RNAi可有效抑制細胞株Ang之表現,但Ang對鼠胚體內發育之生理功能或注射U6-ANG1 RNAi表現卡匣對著床後鼠胚發育之影響,仍需進一步之研究。

RNA interference (RNAi), mediated by double-stranded RNA (dsRNA), has been demonstrated being capable of silencing gene expression in a sequence-specific manner. It has been applied to study the function of genes interested. Mouse angiogenin (Ang) is a constitutive protein in the blood and RNase A gene family including Angiogenin, RNase 1 and RNase 4 are located on mouse chromosome 14. The aim of this study was to investigate the effect of Ang expression on the development of mouse embryos by RNAi. In Experiments 1, the expression patterns of RNase A gene family in the pre-/postimplantation mouse embryos and different mouse cell lines were examined by RT-PCR. The expression of Ang was detected at E1.5, E2.5 and E3.5, E7.5, E9.5 and E12.5 mouse embryos/fetuses. RNase 4 was expressed weakly in E7.5 fetus but expressed strongly at the stage beyond. However, RNase 1 could only be detected in E9.5 and E12.5 fetuses. Ang and RNse 1 were detected in mouse CT-26, 3T3 and B16F10 cell lines, but RNase 4 was not expressed in B16F10 cells. After transfection of the three Ang-RNAi expression vectors (pUC-ANG1, -ANG2, -ANG3) into CT-26 and B16F10 cells, the expression of Ang was inhibited significantly (P<0.05). However, different inhibitory efficiencies were observed in different cell lines. In CT26 cells, the expression of Ang was reduced to 22%, 47% and 47% by pUC-ANG1, pUC-ANG2 and pUC-ANG3, respectively, while in B16F10, they were 57%, 18% and 33%. Additionaly, the inhibitory effects of pUC-ANG2 and pUC-ANG3 to Ang expression in B16F10 were significantly higher than pUC-ANG1 was. In Experiment 2, the U6-ANG1 RNAi expression cassette was introduced into the pronucleus of mouse zygotes by microinjection to evaluate the effect of Ang on the development of mouse embryos. Results showed that there was no significant difference on the percentages of blastocyst formation among the control (non-injected), U6-ANG1 injected, and U6-ANG1+EGPF coinjected groups. The percentages of embryos developed to morula stage in the U6-ANG1-injected (65%) and the coinjected groups (58%) were significantly lower than that of the control group (73%). A significant difference was also found between the two gene-injected groups. After transfer of the coinjected embryos into recipient mice, four out of the twenty four pups born were verified as EGFP transgenic mice by PCR, but none of them carried the U6-ANG1 insert. In this study, Ang expression was detected in the pre- and postimplantation mouse embryos And the Ang-RNAi expression vectors could inhibit the expression of Ang in different cell lines. Introduction of siRNA targeting Ang gene expression to mouse zygots might be lethal to the postimplantational development of mouse embryos, which requires further investigation.
Appears in Collections:動物科學系

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