紀(jì)慧麗，盧晟盛，潘登科

1. 廣西大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，亞熱帶農(nóng)業(yè)生物資源保護(hù)與利用國家重點(diǎn)實(shí)驗(yàn)室，南寧 530004；

2. 中國農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所，農(nóng)業(yè)部畜禽遺傳資源與種質(zhì)創(chuàng)新重點(diǎn)實(shí)驗(yàn)室，北京 100193

體細(xì)胞核移植后表觀遺傳重編程的異常及其修復(fù)

紀(jì)慧麗1,2，盧晟盛1，潘登科2

1. 廣西大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，亞熱帶農(nóng)業(yè)生物資源保護(hù)與利用國家重點(diǎn)實(shí)驗(yàn)室，南寧 530004；

2. 中國農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所，農(nóng)業(yè)部畜禽遺傳資源與種質(zhì)創(chuàng)新重點(diǎn)實(shí)驗(yàn)室，北京 100193

體細(xì)胞核移植(Somatic cell nuclear transfer, SCNT)是指將高度分化的體細(xì)胞移入到去核的卵母細(xì)胞中發(fā)育并最終產(chǎn)生后代的技術(shù)。然而，體細(xì)胞克隆的總體效率仍然處于一個(gè)較低的水平，主要原因之一是由于體細(xì)胞供體核不完全的表觀遺傳重編程，包括DNA甲基化、組蛋白乙?；?、基因組印記、X染色體失活和端粒長(zhǎng)度等修飾出現(xiàn)的異常。使用一些小分子化合物以及Xist基因的敲除或敲低等方法能修復(fù)表觀遺傳修飾錯(cuò)誤，輔助供體核的重編程，從而提高體細(xì)胞克隆效率，使其更好地應(yīng)用于基礎(chǔ)研究和生產(chǎn)實(shí)踐。文章對(duì)體細(xì)胞核移植后胚胎發(fā)育過程中出現(xiàn)的異常表觀遺傳修飾進(jìn)行了綜述，并著重論述了近年來有關(guān)修復(fù)表觀遺傳錯(cuò)誤的研究進(jìn)展。

體細(xì)胞核移植；重編程；表觀遺傳異常；小分子化合物

Keywords:SCNT; reprogramming; epigenetic errors; small molecules

體細(xì)胞核移植(Somatic cell nuclear transfer, SCNT)技術(shù)是將高度分化的體細(xì)胞核移植到去核的卵母細(xì)胞中使之構(gòu)成重構(gòu)胚，通過將克隆胚胎移植到代孕動(dòng)物體內(nèi)后獲得與供體細(xì)胞具有相同基因組的克隆動(dòng)物。1997年，Wilmut等[1]首次報(bào)道了以成體綿羊乳腺上皮細(xì)胞為核供體的克隆羊“Dolly”的誕生，證明了高度分化的體細(xì)胞仍具有全能性，激起了各國科學(xué)家對(duì)體細(xì)胞克隆動(dòng)物的研究熱潮。目前，通過體細(xì)胞核移植技術(shù)已經(jīng)成功獲得了多種哺乳動(dòng)物的克隆后代[1～6]。 然而，體細(xì)胞克隆胚胎的發(fā)育能力低，只有一部分的重構(gòu)胚胎能發(fā)育到足月，其中有許多在出生后不久就死亡[6,7]，即使存活的后代也表現(xiàn)出胎盤肥大[8,9]、胎兒過度生長(zhǎng)[10]等，這種現(xiàn)象稱為“巨胎征”。

造成克隆效率低以及克隆動(dòng)物各類異常表型的主要原因之一是表觀遺傳修飾在克隆胚胎早期發(fā)育階段的異常重編程[11]。體細(xì)胞核在卵胞質(zhì)中發(fā)生重新編程，轉(zhuǎn)變?yōu)橐环N全能的胚胎狀態(tài)，這其中包含了復(fù)雜的表觀遺傳修飾，包括染色質(zhì)重塑、DNA甲基化、組蛋白共價(jià)修飾、X染色體失活和印記基因表達(dá)等。體細(xì)胞重編程在哺乳動(dòng)物和克隆胚胎的發(fā)育過程中，對(duì)建立核的全能性起關(guān)鍵作用。研究證明，供體細(xì)胞核的不完全重編程造成錯(cuò)誤的表觀遺傳，引起相關(guān)基因的異常表達(dá)，最終影響克隆胚胎的發(fā)育[12]。為了提高克隆動(dòng)物的制備效率，科研人員嘗試多種方法來提高克隆效率，例如改良去核程序[13]、優(yōu)化重構(gòu)胚的激活[14]、供體細(xì)胞的化學(xué)處理[15]、連續(xù)兩次核移植[16]，但是與正常受精的胚胎相比，克隆動(dòng)物制備效率仍然很低，而且在不同物種中存在很大的差異。本文主要對(duì)克隆胚胎中錯(cuò)誤的表觀遺傳修飾以及如何修復(fù)進(jìn)行了綜述。

1 體細(xì)胞核移植過程中異常的表觀遺傳修飾

1.1 DNA甲基化

體細(xì)胞克隆動(dòng)物普遍存在 DNA甲基化水平過高的現(xiàn)象，主要表現(xiàn)在去甲基化不充分和提前甲基化?？寺∨Ｅ咛サ幕蚪M去甲基化發(fā)生時(shí)間跟正常胚胎幾乎相同，在 2-細(xì)胞期達(dá)到最低，隨后出現(xiàn)過早重新甲基化，甲基化水平上升；體細(xì)胞基因組在核移植后基因組去甲基化的程度較受精胚胎低，其DNA甲基化水平明顯高于正常胚胎的水平而更接近于體細(xì)胞狀態(tài)[17]。Dean等[18]檢測(cè)克隆牛桑椹胚處于細(xì)胞分裂中期的細(xì)胞核，發(fā)現(xiàn)了較高的甲基化水平，在4～8細(xì)胞期重新甲基化提前發(fā)生，這可能是因?yàn)轶w細(xì)胞核中甲基化酶的存在形式和卵中的不同，且滋養(yǎng)層細(xì)胞發(fā)生異常的超甲基化。在小鼠克隆胚胎，核移植后囊胚期重新甲基化之前發(fā)生持續(xù)的去甲基化，滋養(yǎng)層細(xì)胞上出現(xiàn)異常的超甲基化修飾。Beaujean等[19]用同樣的方法分析綿羊成纖維細(xì)胞核移植胚胎，發(fā)現(xiàn)2～8細(xì)胞期的核移植胚胎染色特征與供體細(xì)胞相似，顯示出過高的甲基化水平，囊胚期滋養(yǎng)層細(xì)胞甲基化程度也較高。總之，克隆胚胎滋養(yǎng)層細(xì)胞的甲基化過高，明顯異常。

1.2 組蛋白修飾

組蛋白乙?；怯山M蛋白乙酰基轉(zhuǎn)移酶(Histone acetyltransferase, HAT)和組蛋白去乙?；?Histone deacetylase, HDAC)協(xié)調(diào)催化的，一般修飾N-末端保守的賴氨酸殘基部位，組蛋白乙?；绊懭旧w中組蛋白和染色質(zhì)之間的相互作用，利于轉(zhuǎn)錄因子結(jié)合和/或可調(diào)節(jié)組蛋白 N-端區(qū)與蛋白質(zhì)之間的相互作用，從而促進(jìn)轉(zhuǎn)錄[20]。研究發(fā)現(xiàn)，克隆牛和水牛胚胎與體外受精胚胎相比，乙?；斤@著降低[21,22]。檢測(cè)克隆豬胚胎發(fā)現(xiàn)，H3K9、H3K14、H4K16位點(diǎn)的乙?；皆隗w細(xì)胞核移入后下降，之后逐漸消失，重構(gòu)胚胎激活后，乙?；幕謴?fù)與孤雌激活胚胎相似，H3/K9、K14乙?；l(fā)生在后/末期，H4K16乙?；l(fā)生在原核期[23]。Wang等[24]研究發(fā)現(xiàn)，小鼠體細(xì)胞核移植后組蛋白H3K9和H3K14高度乙?；?，隨后核質(zhì)進(jìn)行重構(gòu)時(shí)乙?；街饾u降低，而且重構(gòu)胚的乙?；w水平比正常受精胚胎低。以上研究表明，體細(xì)胞核移植胚胎中，組蛋白乙酰化的重編程是不完全的。

組蛋白甲基化修飾主要發(fā)生在賴氨酸和精氨酸側(cè)鏈，較組蛋白乙?；鼮閺?fù)雜，可發(fā)生一甲基化、二甲基化和三甲基化修飾。組蛋白 H3K9的三甲基化和二甲基化與基因的沉默相關(guān)，而 H3K4甲基化引起基因轉(zhuǎn)錄的起始[25]。研究發(fā)現(xiàn)，克隆小鼠胚胎在激活后 H3K9的三甲基化和二甲基化逐漸地去甲基化，與正常受精胚胎不同，克隆胚胎中沒有觀察到H3K9三甲基化或二甲基化的不對(duì)稱分布[24]。同樣，Zhang等[26]也發(fā)現(xiàn)了克隆胚胎中異常的組蛋白甲基化修飾，正常囊胚的內(nèi)細(xì)胞團(tuán)中H3K27是三甲基化，而在克隆囊胚中卻不存在這種修飾。

1.3 X染色體失活

XIST基因在 X染色體失活(X chromosome inactivation, XCI)中起重要作用，其編碼非翻譯的RNA ，啟動(dòng)X染色體的失活，XIST基因在X染色體的失活中心開始轉(zhuǎn)錄，并使整條染色體轉(zhuǎn)錄失活。在沒有XIST RNA的情況下，X染色體失活也能被起始，但XIST是X染色體維持穩(wěn)定沉默所必需的[27]。研究發(fā)現(xiàn)，在克隆小鼠囊胚中，失活的X染色體被重新激活，但在后期發(fā)育中X連鎖基因異常表達(dá)，沒有顯示 XCI印記的正常模式[28]。Inoue等[29]研究發(fā)現(xiàn)，在雌性和雄性克隆胚胎中，XIST的表達(dá)水平顯著高于對(duì)照組體外受精胚胎，XIST的異常表達(dá)首先出現(xiàn)在 4-細(xì)胞期，在囊胚期增強(qiáng)。流產(chǎn)克隆牛胎兒和死亡初生牛犢內(nèi)XCI的模式發(fā)生了改變，有胎盤的牛表現(xiàn)出X染色體隨機(jī)失活，與正常對(duì)照組和健康克隆小牛中優(yōu)先失活父源X染色體相反[30]。在克隆牛囊胚中沒有發(fā)現(xiàn)X連鎖基因的異常表達(dá)，但是在后期發(fā)育中發(fā)現(xiàn)，死亡克隆牛的胎盤中X連鎖基因是雙等位基因表達(dá)，而不是母本特異性表達(dá)[31]，然而在存活的克隆牛胎盤中只有一條未失活的X染色體[30]，這表明XCI的異常模式導(dǎo)致了胎兒的死亡，X連鎖基因的異常表達(dá)嚴(yán)重影響胎盤的發(fā)育。

1.4 基因組印記

基因組印記(Genomic imprinting)指控制某一表型的一對(duì)等位基因由于親源不同而差異性表達(dá)，即機(jī)體只表達(dá)來自親本一方的等位基因。基因組印記的產(chǎn)生及其控制未涉及到 DNA 序列的變化，主要依靠 DNA 差異甲基化等非基因突變因素維持?；虻恼Ｓ∮泴?duì)哺乳動(dòng)物胚胎和胎兒的正常發(fā)育極其重要，錯(cuò)誤的印記會(huì)引起嚴(yán)重的遺傳疾病或致癌。在克隆動(dòng)物中觀察到最普遍的表型是胎兒生長(zhǎng)異常，例如胎盤肥大、出生體重增大以及胎兒死亡，這是天然存在和印記基因的定向突變共同的作用結(jié)果，表明印記基因的異常表達(dá)可能會(huì)導(dǎo)致克隆動(dòng)物異常發(fā)育[32]。在克隆小鼠中，沒有觀察到任何單一印記基因的異常表達(dá)與異常胎兒過度生長(zhǎng)的程度之間有實(shí)質(zhì)性的關(guān)聯(lián)，然而，幾個(gè)印記基因的累積失調(diào)對(duì)胎兒的生長(zhǎng)具有相對(duì)的影響[33]。分析發(fā)現(xiàn)，植入前胚胎發(fā)育相關(guān)的印記基因——胰島素樣生長(zhǎng)因子-2(Igf2)在克隆胚胎中的表達(dá)顯著高于體外受精胚胎[34]，Igf2的過表達(dá)和Igf2r的表達(dá)下調(diào)導(dǎo)致小鼠胎兒和胎盤的過度生長(zhǎng)[35]。在死亡克隆牛器官內(nèi) Igf2和H19基因表達(dá)均出現(xiàn)異常，而在成活的克隆牛體內(nèi)，只有肌肉內(nèi)的Igf2基因表達(dá)變化較大[36]。同樣，在死亡克隆豬胎盤上發(fā)現(xiàn)Igf2和H19基因呈現(xiàn)異常的高甲基化水平[37]。總之，體細(xì)胞克隆過程中印記基因的異常表達(dá)是引起克隆后代發(fā)育異常和死亡的原因之一。

1.5 端粒長(zhǎng)度

端粒是線狀染色體的天然末端，通過阻止末端編碼DNA序列的損耗和防止端-端染色體融合，在維持染色體 DNA的完整性方面發(fā)揮關(guān)鍵作用[38]。一般來說，端粒長(zhǎng)度的一些損失發(fā)生在每個(gè)細(xì)胞分裂時(shí)，是滯后鏈不完全復(fù)制的結(jié)果。多莉羊是由成年上皮細(xì)胞做供體克隆所得，其端粒比同齡自然繁育羊短[39]。然而，后來的研究發(fā)現(xiàn)，克隆動(dòng)物的端粒長(zhǎng)度與同齡自然繁育的對(duì)照組相似，克隆動(dòng)物中端粒酶的活性被重新激活到與對(duì)照組相似的水平[40]。端粒長(zhǎng)度的調(diào)節(jié)在一定程度上與用于克隆的供體細(xì)胞類型有關(guān)，由成纖維細(xì)胞或肌細(xì)胞得到的克隆牛的端粒長(zhǎng)度與年齡相仿的對(duì)照組相似，然而上皮細(xì)胞得到的克隆牛的端粒沒有恢復(fù)到正常長(zhǎng)度[41]。在克隆桑椹胚期端粒處于供體細(xì)胞的水平，而囊胚期端粒已經(jīng)恢復(fù)到正常長(zhǎng)度，這可能是由于早期胚胎中的端粒酶重新調(diào)整了供體基因組縮短的端粒[42]。

2 修復(fù)異常的表觀遺傳修飾

體細(xì)胞核移植技術(shù)效率低下的主要原因是分化的體細(xì)胞核不能被卵母細(xì)胞質(zhì)完全重編程，主要表現(xiàn)為異常的表觀遺傳修飾模式。如何修復(fù)這些異常的表觀遺傳修飾，目前的研究大多是使用能改變表觀遺傳水平的小分子化合物處理重構(gòu)胚胎，以及敲除或敲低相關(guān)基因來改變表觀遺傳修飾。

2.1 DNA甲基化抑制劑

DNA甲基化被兩種 DNA甲基轉(zhuǎn)移酶(DNA methyltransferase, DNMT)催化：DNMT1維持DNA復(fù)制期間已經(jīng)建立的甲基化模式，而 DNMT3a和DNMT3b參與建立胚胎植入前的從頭甲基化模式。通過應(yīng)用DNA甲基轉(zhuǎn)移酶抑制劑(DNMTi)，可以抑制異常DNA甲基化的發(fā)生，修復(fù)表觀遺傳錯(cuò)誤，從而提高動(dòng)物克隆效率。研究證明，S-腺苷高半胱氨酸(S-adenosylhomocysteine, SAH)[43]、5-氮雜-2′-脫氧胞苷(5-aza-dC)[21,44]和RG108[45]能改變DNA甲基化修飾。

SAH是一種非核苷類化合物DNMTi，沒有細(xì)胞毒性，是S-腺苷甲硫氨酸(SAM)代謝產(chǎn)物的類似物，而SAM是DNA甲基化的主要甲基供體，SAH能抑制細(xì)胞內(nèi)大多數(shù)甲基轉(zhuǎn)移酶的活性而并不摻入DNA。用SAH處理供體細(xì)胞成纖維細(xì)胞，發(fā)現(xiàn)SAH能誘導(dǎo)總體 DNA去甲基化，并且能提高端粒酶活性、重新激活部分失活的X染色體，所得到的克隆胚胎的發(fā)育潛力有所提高[43]。

5-aza-dC是一種核苷類似物 DNMTi，主要在DNA復(fù)制過程中摻入DNA，然后被DNA甲基轉(zhuǎn)移酶識(shí)別，并與其共價(jià)結(jié)合而抑制該酶的活性，使基因組DNA發(fā)生去甲基化。Enright等[21]用0.01 μmol/L的5-aza-dC處理牛供體細(xì)胞降低了 DNA甲基化水平，同時(shí)組蛋白乙?；揭灿兴岣?。5-aza-dC和TSA聯(lián)合處理供體細(xì)胞和克隆胚胎，顯著降低了DNA甲基化水平，提高了克隆胚胎的體外發(fā)育潛力[44]。

RG108是一種新型的色氨酸衍生物類小分子化合物DNMTi，細(xì)胞毒性小，通過與DNMT的活性位點(diǎn)非共價(jià)結(jié)合，阻礙其余 DNA結(jié)合，從而降低DNA甲基化水平。Xu等[45]發(fā)現(xiàn)RG108和Scriptaid聯(lián)合使用能顯著促進(jìn)克隆胚胎中 NANOG基因的轉(zhuǎn)錄，增強(qiáng)植入前胚胎的發(fā)育潛力，修復(fù)體細(xì)胞克隆中H19的ICR3位點(diǎn)破裂的印跡DNA甲基化。

2.2 組蛋白去乙?；敢种苿?/p>

組蛋白去乙酰化酶(HDAC)一般可以分為 5種類型：ClassⅠ(HDAC1,3和8)，ClassⅡa(HDAC4,5,7和9)，ClassⅡb(HDAC6和10)，ClassⅢ(SIRT1到7)和 ClassⅣ(HDAC11)。TSA、Scriptaid、SAHA和Oxamflatin能抑制 ClassⅠ和 ClassⅡa/b HDAC；APHA能抑制ClassⅠ和ClassⅡa/b，尤其是HDAC3和HDAC6；VPA能抑制ClassⅠ和ClassⅡa；Sirtinol只能抑制ClassⅢ HDAC[46]。

曲古抑菌素A(Trichostatin A, TSA)是第一種被發(fā)現(xiàn)對(duì)克隆動(dòng)物制備有效的小分子化合物，TSA抑制 HDAC的作用從而使染色質(zhì)處于高度乙?；癄顟B(tài)，這樣有利于轉(zhuǎn)錄因子的結(jié)合，使基因處于轉(zhuǎn)錄激活狀態(tài)。研究發(fā)現(xiàn)在牛[47,48]、豬[49]、兔[50]和小鼠[51]中，用HDAC抑制劑TSA處理核移植供體細(xì)胞，可以提高克隆胚胎的體外發(fā)育率。用TSA處理重構(gòu)胚可以提高胚胎中H3K14、H4K12乙?；剑M(jìn)而提高克隆效率[37]。TSA處理小鼠克隆胚胎后進(jìn)行連續(xù)克隆，已經(jīng)獲得了25代約500只克隆小鼠，而且克隆效率并沒有逐漸下降[52]。然而，TSA對(duì)克隆胚胎有毒性作用，其增強(qiáng)克隆胚胎的重編程的效果取決于不同種系的供體細(xì)胞對(duì)藥物的敏感度[53]。

Scriptaid是一種人工合成的低毒性HDAC抑制劑，可誘導(dǎo)組蛋白過乙?；鹑旧w重建，增強(qiáng)細(xì)胞內(nèi)的轉(zhuǎn)錄活性和蛋白質(zhì)的表達(dá)。Van Thuan等[54]利用250 nmol/L Scriptaid處理近交系小鼠克隆胚胎10 h，提高了克隆胚胎的發(fā)育能力，同時(shí)成功獲得克隆后代，結(jié)束了近交品系小鼠無法克隆的歷史。

Ono等[46]發(fā)現(xiàn)，SAHA和Oxamflatin能降低囊胚細(xì)胞的細(xì)胞凋亡水平，提高克隆小鼠發(fā)育到足月，并能顯著提升核移植胚胎獲得的胚胎干細(xì)胞(ntES)的建系效率，而且不會(huì)導(dǎo)致明顯的異常。Su等[55]研究發(fā)現(xiàn)，Oxamflatin修改了H3K9和H3K18的乙?；癄顟B(tài)，提高了囊胚的質(zhì)量，并且顯著提高了克隆牛胚胎的體外發(fā)育。

丙戊酸(Valproic acid，VPA)是一種短鏈脂肪酸，一直以來被用于治療癲癇，目前發(fā)現(xiàn)VPA可以誘導(dǎo)分化細(xì)胞的重編程。Miyoshi等[56]發(fā)現(xiàn)VPA能夠提高小型豬克隆胚胎的體外發(fā)育以及Oct3/4基因的表達(dá)。用4 mmol/L的VPA處理24 h，能顯著提高克隆牛胚胎的發(fā)育，提高了 H3K9的乙?；剑蚨鰪?qiáng)了細(xì)胞核重編程[57]。Kim等[58]研究發(fā)現(xiàn)VPA比TSA更有效地提高克隆胚胎的發(fā)育，然而，VPA對(duì)提高小鼠的克隆效率的作用甚微，但卻能改善小鼠 SCNT胚胎中Oct4基因的表達(dá)以及 K3K27me3的核分布[59]。

Jin等[60]發(fā)現(xiàn)，一種新型的廣譜 HDAC抑制劑LBH589(Panobinostat)通過改變表觀遺傳狀態(tài)和基因表達(dá)增強(qiáng)核重編程以及SCNT胚胎的發(fā)育潛力，并能提高囊胚的質(zhì)量。后來，他們又研究發(fā)現(xiàn)，1 μmol/L的CUDC-101處理24 h后能顯著提高豬SCNT胚胎的發(fā)育能力，免疫熒光檢測(cè)發(fā)現(xiàn)，H3K9的乙?；礁哂趯?duì)照組[61]。

Song等[62]研究發(fā)現(xiàn)，一種極性雜合化合物CBHA(m-carboxycinnamic acid bishydroxamide)能提高體細(xì)胞克隆豬胚胎的體外發(fā)育能力，提高總體的組蛋白乙?；?，并能修正早期發(fā)育的一些重要基因的表達(dá)。Dai等[63]先前也報(bào)道了CBHA 能提高小鼠克隆胚胎的發(fā)育能力，并能提高SCNT胚胎建立胚胎干細(xì)胞系的效率。

雖然HDAC抑制劑處理能提高克隆效率，但出生動(dòng)物的存活效率并沒有提高，而且HDAC抑制劑處理提高克隆效率的機(jī)制還不是很清楚。但可以推測(cè)，HDAC抑制劑可引起核心組蛋白的過乙?；?，導(dǎo)致染色質(zhì)結(jié)構(gòu)松弛，能夠更容易結(jié)合轉(zhuǎn)錄因子，還能使供體細(xì)胞的基因組在核移植后發(fā)生 DNA去甲基化。有報(bào)道顯示，用HDAC抑制劑處理能夠提高組蛋白乙?；?、初期mRNA的產(chǎn)生和基因表達(dá)，與正常受精胚胎的狀態(tài)相似[64]。

2.3 敲低或敲除Xist基因

對(duì)克隆囊胚基因表達(dá)譜的分析發(fā)現(xiàn)，與體外受精囊胚相比，克隆胚胎X染色體上許多基因的特異性表達(dá)下調(diào)[29]。X連鎖基因的這種表達(dá)抑制與 Xist基因的表達(dá)升高相關(guān)聯(lián)，而Xist是雌性細(xì)胞中負(fù)責(zé)失活其中一條X染色體，RNA原位雜交熒光分析卵裂球細(xì)胞核中Xist的mRNA，雄性和雌性克隆胚胎中顯示一個(gè)過度的信號(hào)，這表明未失活的X染色體上Xist基因的表達(dá)異常[64]。當(dāng)使用Xist敲除的供體細(xì)胞用于核移植時(shí)，卵丘細(xì)胞和支持細(xì)胞的克隆出生率分別升高了8倍和14倍[29]。同樣，通過向重構(gòu)卵母細(xì)胞中注射干擾RNA來敲低Xist，克隆出生率提高了大約10倍[65]。因此，敲除或敲低基因可能部分修復(fù)X連鎖基因的異常表達(dá)，從而提高克隆效率。

3 展 望

體細(xì)胞核移植技術(shù)具有重要的理論研究和生產(chǎn)價(jià)值，可應(yīng)用于治療性克隆、瀕臨滅絕品種的保護(hù)、家畜的品種改良及大量繁殖、轉(zhuǎn)基因動(dòng)物生產(chǎn)、人類器官移植和建立疾病模型等研究。然而，隨著誘導(dǎo)多能性胚胎干細(xì)胞(Induced pluripotent stem cells, iPS)技術(shù)的出現(xiàn)，人們的研究重點(diǎn)轉(zhuǎn)向了 iPS 研究。但是許多研究已經(jīng)證實(shí)，iPS 技術(shù)誘導(dǎo)獲得的 iPS細(xì)胞質(zhì)量不如體細(xì)胞核移植細(xì)胞，目前僅有小鼠的iPS 細(xì)胞能完全發(fā)育為個(gè)體[66,67]，而體細(xì)胞核移植技術(shù)能夠?qū)⒔^大多數(shù)物種分化的體細(xì)胞重編程為多能性干細(xì)胞并發(fā)育為個(gè)體。因此，體細(xì)胞核移植技術(shù)仍是研究核重編程機(jī)制的一種重要技術(shù)手段。

表觀遺傳修飾是核重編程的關(guān)鍵，研究發(fā)現(xiàn)DNA甲基轉(zhuǎn)移酶抑制劑和組蛋白去乙酰化酶抑制劑等能部分修復(fù)克隆胚胎異常的表觀遺傳修飾，能提高供體核的重編程。然而，目前使用的大多數(shù)試劑都有一定的毒副作用，因此，進(jìn)一步研究效率高、毒副作用小的藥物來修復(fù)表觀遺傳錯(cuò)誤將是今后的主要發(fā)展方向之一。核移植后基因印記發(fā)生異常是引起克隆動(dòng)物死亡的重要原因之一。目前，關(guān)于如何防止體細(xì)胞核移植后基因印記發(fā)生異常的研究較少，Hikichi等[68]利用孤雌胚胎的胚胎干細(xì)胞進(jìn)行核移植，發(fā)現(xiàn)克隆小鼠的胎盤中印記基因Peg10的表達(dá)接近正常水平，說明不同來源的供核細(xì)胞對(duì)克隆動(dòng)物的印記基因重編程有一定的影響。

新技術(shù)的應(yīng)用如高通量測(cè)序技術(shù)有助于發(fā)現(xiàn)克隆胚胎中基因的異常表達(dá)[69]。最近，利用轉(zhuǎn)錄組分析發(fā)現(xiàn)H3K9me3是SCNT有效重編程的一個(gè)主要障礙，除去H3K9me3能顯著提高克隆效率[70]。總之，隨著對(duì)表觀遺傳重編程的深入研究，體細(xì)胞核移植技術(shù)將能更好地應(yīng)用于基礎(chǔ)研究和生產(chǎn)實(shí)踐。

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(責(zé)任編委: 方向東)

Epigenetic reprogramming by somatic cell nuclear transfer: questions and potential solutions

Huili Ji1,2, Shengsheng Lu1, Dengke Pan1

1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
2. Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation, Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Somatic cell nuclear transfer (SCNT) is a technology by which a highly differentiated somatic nucleus is transferred into an enucleated oocyte to generate a reconstructed embryo that subsequently develops to an offspring. However, to date, the efficiency of cloned animal is still low. The major reason is incomplete nuclear reprogramming of donor cells after nuclear transfer, which results in abnormal epigenetic modifications, including DNA methylation, histone acetylation, gene imprinting, X-chromosome inactivation, and telomere length. Most improvements have been made in somatic epigenetic reprogramming with small molecules and manipulating expression of specific genes. It is expected that SCNT will soon have broad applications in both basic research and practical production. In this review, we summarize the recent progress in epigenetic reprogramming by somatic cell nuclear transfer; in particular, we focus on strategies for rescuing the epigenetic errors occurring during SCNT.

2014-07-17;

2014-09-19

轉(zhuǎn)基因生物新品種培育重大專項(xiàng)(編號(hào)：2014ZX0800605B)和國家高技術(shù)研究發(fā)展計(jì)劃(863計(jì)劃)項(xiàng)目(編號(hào)：2012AA020601)資助

紀(jì)慧麗，碩士研究生，專業(yè)方向：動(dòng)物胚胎生物技術(shù)。E-mail: jihuili1989@163.com

潘登科，博士，副研究員，研究方向：動(dòng)物胚胎生物技術(shù)。E-mail: pandengke2002@163.com

盧晟盛，博士，研究員，博士生導(dǎo)師，研究方向：動(dòng)物繁殖生物技術(shù)。E-mail: sslu@gxu.edu.cn

10.3724/SP.J.1005.2014.1211

時(shí)間: 2014-10-10 16:09:38

URL: http://www.cnki.net/kcms/detail/11.1913.R.20141010.1609.001.html