山羊克隆胎兒不同組織中H19基因CpG島甲基化模式及表達(dá)量檢測(cè)

李長(zhǎng)雷，鄭聰穎，劉軍，蘭杰，李文哲，張涌

西北農(nóng)林科技大學(xué)動(dòng)物醫(yī)學(xué)院 農(nóng)業(yè)部動(dòng)物生殖生理與胚胎工程重點(diǎn)實(shí)驗(yàn)室，楊凌 712100

動(dòng)物及獸醫(yī)生物技術(shù)

山羊克隆胎兒不同組織中H19基因CpG島甲基化模式及表達(dá)量檢測(cè)

李長(zhǎng)雷，鄭聰穎，劉軍，蘭杰，李文哲，張涌

西北農(nóng)林科技大學(xué)動(dòng)物醫(yī)學(xué)院 農(nóng)業(yè)部動(dòng)物生殖生理與胚胎工程重點(diǎn)實(shí)驗(yàn)室，楊凌 712100

表觀重編程異常是核移植胚胎發(fā)育異常的重要原因。為了研究克隆山羊胎兒不同組織中H19基因CpG島甲基化水平和相對(duì)表達(dá)量，本實(shí)驗(yàn)運(yùn)用亞硫酸鹽法和熒光實(shí)時(shí)定量PCR法分別檢測(cè)了死亡克隆山羊胎兒和同期普通山羊胎兒 (對(duì)照組) 肝臟、胎盤(pán)、腎臟、肺臟和心臟組織中H19基因CpG島甲基化水平和mRNA的相對(duì)表達(dá)量。結(jié)果表明，克隆山羊胎兒胎盤(pán)組織中H19基因第5個(gè)CpG島的甲基化水平顯著高于對(duì)照組 (70%vs49.41%，P＜0.05)，H19基因相對(duì)表達(dá)量顯著低于對(duì)照組 (883.3vs1 264.5，P＜0.05)；肺臟組織甲基化水平顯著低于對(duì)照組 (63.53%vs88.24%，P＜0.05)，相對(duì)表達(dá)量顯著高于對(duì)照組 (1 003.4vs515.5，P＜0.05)；其他各組差異不顯著 (P＞0.05)。結(jié)果說(shuō)明，H19基因在克隆山羊胎兒部分組織中 DNA甲基化重編程異常，而且這種異常影響H19基因的正常表達(dá)，這也可能是導(dǎo)致克隆動(dòng)物死亡的重要因素之一。

克隆，山羊，H19基因，CpG島，甲基化模式，mRNA表達(dá)

Abstract:The aberrant epigenetic reprogramme is an important cause for abnormal development of nuclear transfer embryos. The objective of this study was to investigate the CpG island methylation profiles and relative expression levels ofH19gene in different tissues of cloned goat fetus. We detected liver, placenta, kidney, lung and heart in the dead cloned goat fetus and the age-matched normal goat fetus (control) by using bisulfite sequencing and real time PCR. Results indicated that methylation levels of the fifth CpG island ofH19gene in dead cloned goat fetus was significant high compared with that in the control in placenta (70%vs49.41%,P< 0.05), and relative expression levels ofH19gene was significant low compared with that in the control (883.3vs1 264.5,P<0.05). Reversely, the methylation levels was significant low compared with that in the control in lung (63.53%vs88.24%,P< 0.05),and relative expression levels was significant high compared with that in the control (1 003.4vs515.5,P< 0.05). The differences of others groups were insignificant (P> 0.05). Results showed the abnormal DNA methylation proflies ofH19gene occurred in some tissues of cloned goat fetus, which affected normal expression levels ofH19gene, indicating that aberrant DNA methylationreprogramme may be one of the important factors for the death of cloned animals.

Keywords:clone, goat,H19gene, CpG island, methylation pattern, mRNA expression

自1996年Campbell等成功獲得第一例體細(xì)胞核移植綿羊以來(lái)，體細(xì)胞克隆技術(shù)已成功應(yīng)用于多種哺乳動(dòng)物，但是通過(guò)核移植技術(shù)得到健康克隆動(dòng)物的效率非常低，體細(xì)胞核移植動(dòng)物的流產(chǎn)率和新生兒死亡率遠(yuǎn)高于體外受精和人工受精得到的動(dòng)物，即使是存活下來(lái)的克隆動(dòng)物也常伴有表型異常和不同程度的發(fā)育缺陷，研究表明表觀重編程異常是核移植胚胎發(fā)育異常的重要原因之一[1-3]。……