體細(xì)胞克隆豬發(fā)育異常研究進(jìn)展

敖政，陳祥，吳珍芳，李紫聰

體細(xì)胞克隆豬發(fā)育異常研究進(jìn)展

敖政1，陳祥1，吳珍芳2，李紫聰2

1. 貴州大學(xué)動物科學(xué)學(xué)院，高原山地動物遺傳育種與繁殖教育部重點(diǎn)實(shí)驗(yàn)室，貴陽 550025 2. 華南農(nóng)業(yè)大學(xué)動物科學(xué)學(xué)院，國家生豬種業(yè)工程研究中心，廣州 510642

克隆又稱體細(xì)胞核移植(somatic cell nuclear transfer, SCNT)，是一種將已分化的細(xì)胞重編程恢復(fù)全能性而生產(chǎn)與供體細(xì)胞基因型完全相同后代的無性繁殖技術(shù)。豬的克隆技術(shù)具有重要的應(yīng)用價(jià)值，包括擴(kuò)繁優(yōu)良種豬、制備基因修飾豬、保護(hù)珍貴和瀕危豬種以及研究豬體細(xì)胞重編程機(jī)制。然而，克隆豬存在出生率和初生重低以及死胎率、新生期死亡率和畸形率高等問題，這些都嚴(yán)重影響了克隆豬的應(yīng)用前景。供體核的表觀重編程錯(cuò)誤被認(rèn)為是克隆效率低和胚胎發(fā)育異常的主要原因，但是目前大多數(shù)研究通過修正表觀重編程錯(cuò)誤并沒有大幅度提高克隆豬的出生率和健康率。本文綜述了克隆豬的異常表型、發(fā)育異常的原因以及提高豬克隆效率的有效方法，以期為提高克隆豬的成活率提供參考。

體細(xì)胞核移植；克隆豬；表觀重編程；發(fā)育異常

體細(xì)胞核移植(somatic cell transfer nuclear, SCNT)是將已分化的細(xì)胞重編程恢復(fù)全能型而生產(chǎn)與供體細(xì)胞基因型完全相同后代的一種無性繁殖技術(shù)[1]。從1996年克隆羊“多莉”誕生至今，多種哺乳動物的體細(xì)胞克隆都相繼取得成功，特別是2018年初，中國科學(xué)家宣布成功獲得了存活的體細(xì)胞克隆猴()，這是體細(xì)胞克隆史上的一次重大突破[2]。豬() SCNT技術(shù)在農(nóng)業(yè)、生物醫(yī)學(xué)和基礎(chǔ)研究領(lǐng)域具有重要的應(yīng)用價(jià)值，包括擴(kuò)繁優(yōu)良種豬、制備基因修飾豬、保護(hù)珍貴和瀕危豬種以及研究豬體細(xì)胞重編程機(jī)制[3,4]。然而，如表1所示，克隆豬的出生率低(只有1%左右)、死胎率高(17%~32.8%)，且存在新生期死亡率高(48.0%~ 74.5%)、畸形率高(29.5%~60.0%)和初生重低等問題[5~19]，這些都嚴(yán)重影響了克隆豬的應(yīng)用前景。供體核的表觀重編程錯(cuò)誤被認(rèn)為是克隆效率低和胚胎發(fā)育異常的主要原因，但是目前大多數(shù)研究通過修正表觀重編程錯(cuò)誤并沒有大幅度提高克隆豬的出生率和健康率。因此，本文綜述了克隆豬的異常表型、發(fā)育異常的原因以及提高豬克隆效率的有效方法，以期為提高克隆豬的成活率提供參考。

1 克隆豬發(fā)育異常

1.1 異常表型

Schmidt等[18]對815頭克隆及轉(zhuǎn)基因豬從出生至斷奶進(jìn)行了跟蹤記錄，結(jié)果發(fā)現(xiàn)新生克隆豬的死亡率為48%，畸形率為29.5%，尸檢結(jié)果顯示死亡克隆豬的主要生理缺陷分布在消化系統(tǒng)、循環(huán)系統(tǒng)、繁殖系統(tǒng)和骨骼肌系統(tǒng)，且存在諸多未發(fā)現(xiàn)的細(xì)微生理缺陷，這些生理缺陷嚴(yán)重影響機(jī)體正常的生理活動，是造成新生克隆豬死亡的直接或間接原因。因此，目前很難確定克隆豬死亡的具體機(jī)制，可能是一種或多種缺陷引發(fā)的結(jié)果。此外，很多新生克隆豬是無征兆的突然死亡，早期研究發(fā)現(xiàn)腦膜炎和血運(yùn)障礙可能是仔豬突然死亡的主要原因[6]?？寺∽胸i因屈肌腱和肘關(guān)節(jié)骨骼發(fā)育不良而導(dǎo)致仔豬站立失敗，且新生克隆豬會表現(xiàn)出不佳的哺乳反射，因此需要人工及時(shí)喂養(yǎng)充足的初乳以增加仔豬的存活率[6]。

表1 代孕母豬懷孕率及克隆效率

— 表示參考文獻(xiàn)無相應(yīng)數(shù)據(jù)；克隆效率等于出生總仔數(shù)與移植胚胎數(shù)的比值。

克隆新生兒呼吸窘迫是克隆動物圍產(chǎn)期死亡的原因之一，其病理特征包括不完整的肺擴(kuò)張、肺泡塌陷、肺泡壁增厚、肺內(nèi)表面活性劑穩(wěn)態(tài)紊亂和透明膜異常，表明克隆新生兒的肺泡功能不全而造成呼吸窘迫[20,21]?？寺∝i具有如肺動脈移位、肺畸形和肺發(fā)育不全等先天缺陷，這些都可能是仔豬圍產(chǎn)期死亡的原因[18]。Park等[22]比較分析了出生后死亡及存活一個(gè)月克隆豬與同齡人工授精(artificial insemination, AI)豬肺臟的基因表達(dá)模式，在出生后死亡克隆豬中鑒定出121個(gè)差異表達(dá)基因(differen-tially expressed genes, DEGs)，這些DEGs可能與克隆豬肺表面活性劑穩(wěn)態(tài)失調(diào)和糖尿病性腎病等病理癥狀密切相關(guān)；在存活克隆豬中鑒定到154個(gè)DEGs，基因功能富集分析發(fā)現(xiàn)這些DEGs與肺泡發(fā)育延遲和MAPK信號通路下調(diào)有關(guān)，表明存活克隆豬仍然有器官功能異常的風(fēng)險(xiǎn)[23]。此外，部分克隆豬患有巨舌，這嚴(yán)重影響到仔豬的攝乳和呼吸，先天和環(huán)境因素導(dǎo)致這類仔豬幾乎不能存活[6,18]。最近，本課題組研究發(fā)現(xiàn)，嘌呤代謝異?？赡苁强寺∝i腎臟發(fā)生病理變化以及新生期死亡的重要原因[24]。

1.2 宮內(nèi)發(fā)育不良

克隆胚胎的形態(tài)和功能異常是克隆豬出生率低的重要原因。豬胚胎在妊娠第10~12天之間經(jīng)歷從球狀到管狀和絲狀的快速轉(zhuǎn)變[25]，Isom等[26]發(fā)現(xiàn)11.3%的豬克隆胚胎在妊娠第14天仍然是球狀，表明部分克隆豬胚胎由于形態(tài)轉(zhuǎn)變失敗而停止發(fā)育?？寺∨咛サ呐弑P(embryonic disc, ED)和滋養(yǎng)層(tro-phectoderm, TE)與正常受精胚胎進(jìn)行轉(zhuǎn)錄組比較，發(fā)現(xiàn)克隆胚胎的ED和TE中都有大量DEGs，其中ED的DEGs主要參與基因表達(dá)的表觀遺傳控制及microRNA介導(dǎo)的基因沉默和細(xì)胞凋亡，TE的DEGs主要與異常的代謝/異化途徑和亞細(xì)胞的組織缺陷有關(guān)[26]。因此，即使胚胎能夠附植成功，但大部分克隆胎兒和胎盤的發(fā)育仍然存在缺陷。Ruan等[16]在對妊娠30天和35天豬克隆胚胎進(jìn)行轉(zhuǎn)錄組研究中發(fā)現(xiàn)，與AI胚胎相比，異?？寺∨咛ブ斜磉_(dá)下調(diào)的基因數(shù)量比正?？寺∨咛ジ?，且大多數(shù)與胚胎發(fā)育相關(guān)的基因在異?？寺∨咛ブ形茨芗せ畋磉_(dá)。此外，大多數(shù)克隆豬具有宮內(nèi)發(fā)育遲緩(intrauterine growth retardation, IUGR)特征，妊娠65天的克隆胎豬體重以及足月初生重顯著低于AI豬[5,19,27]。初生重是一個(gè)重要的新生兒發(fā)病預(yù)測指標(biāo)，克隆豬的初生重低可能是其出生后高頻率死亡的重要原因[5]。

2 克隆豬發(fā)育異常的原因

2.1 表觀重編程錯(cuò)誤

目前，供體核的表觀重編程錯(cuò)誤成為克隆胚胎發(fā)育異常的主流觀點(diǎn)，主要包括DNA甲基化、組蛋白修飾和X染色體失活異常[28]。很多研究團(tuán)隊(duì)已經(jīng)嘗試通過修正這些表觀遺傳修飾錯(cuò)誤來改變克隆胚胎的命運(yùn)。DNA甲基化重塑是早期胚胎發(fā)育的關(guān)鍵步驟，涉及DNA去甲基化和再甲基化，由DNA甲基轉(zhuǎn)移酶(DNA methyltransferase, DNMT)的催化作用完成[29]。供體細(xì)胞是克隆胚胎發(fā)育的起點(diǎn)，其表觀修飾狀態(tài)會直接影響克隆胚胎的發(fā)育能力[30]。研究發(fā)現(xiàn)，骨髓間充質(zhì)干細(xì)胞(bone marrow stroma cell, BMSC)的囊胚率顯著高于胎兒成纖維細(xì)胞(fetal fib-roblasts, FF)，可能由于BMSC來源的胚胎具有更低的和基因啟動子DNA甲基化水平、更高的H3K9Ac水平和更低的H3K9me3和5-甲基胞嘧啶水平[31]。因此，不同類型的供體細(xì)胞的克隆胚胎發(fā)育效率具有明顯差異，這很可能與供體細(xì)胞的分化程度有關(guān)[11,32]。DNA甲基化重塑異常幾乎是所有克隆動物共有的特征，包括去甲基化不完全和再甲基化異常[33,34]。附植前胚胎的DNA甲基化需要經(jīng)歷特定的變化，其中早期和后期囊胚具有相似的DNA甲基化水平，且后期囊胚內(nèi)細(xì)胞團(tuán)(inner cell mass, ICM)的DNA甲基化水平顯著高于TE[34]。然而，早期克隆囊胚的DNA甲基化水平顯著高于后期囊胚，且后期囊胚的ICM與TE的DNA甲基化水平?jīng)]有差異，表明DNA甲基化印記的異常變化可能造成胎兒和胎盤的發(fā)育缺陷[35]。最近，Gao等[36]研究發(fā)現(xiàn)附植前克隆胚胎經(jīng)歷異常DNA再甲基化，通過特定的DNMT抑制劑使DNA甲基化恢復(fù)到正常水平顯著提高了小鼠()的克隆效率，表明異常的DNA再甲基化也是抑制克隆胚胎發(fā)育的重要原因。DNA甲基化異?？赡芘c基因表達(dá)失調(diào)有關(guān)，抑制基因的表達(dá)降低克隆胚胎中異常的高甲基化水平而利于基因的轉(zhuǎn)錄激活。研究發(fā)現(xiàn)，DNMT抑制劑RG108能促進(jìn)DNA的主動和被動去甲基化以及轉(zhuǎn)錄而增強(qiáng)附植前克隆豬胚胎的發(fā)育能力[29,37]。對妊娠中期克隆胎兒流產(chǎn)的研究發(fā)現(xiàn)，胎兒的DNA重復(fù)區(qū)域和衛(wèi)星序列都呈現(xiàn)高度甲基化，且胎兒和胎盤中很多印跡基因表達(dá)異常以及的DMR3處于低甲基化水平，表明胎兒和胎盤發(fā)育異常都可能造成流產(chǎn)[38]。此外，DNA甲基化變化可能與克隆仔豬的異常表型有關(guān)。出生后的異常克隆豬與正常受精豬在全基因組的基因表達(dá)模式和DNA甲基化水平存在明顯差異[39]，且異?？寺∝i的全基因組相對正常克隆豬更多是呈現(xiàn)低DNA甲基化水平[40]。然而，不能定論異常克隆仔豬的全基因組都是處于低DNA甲基化水平，因?yàn)槠銫pG島區(qū)域具有更高的DNA甲基化水平[40]。盡管表型正常的克隆豬與普通豬的基因表達(dá)模式高度相似，但是克隆豬呈現(xiàn)更多不同的單拷貝序列DNA甲基化模式，DNA甲基化水平的差異可能會影響克隆豬的組織或器官發(fā)育[41]。

哺乳動物基因組的組蛋白的N末端有很多修飾形式，包括甲基化、乙?；⒘姿峄头核鼗萚42]。這些修飾可以影響組蛋白與染色質(zhì)的相互作用而調(diào)控基因的轉(zhuǎn)錄[28]。常見的組蛋白修飾是甲基化和乙?；?。組蛋白乙酰化可以減弱組蛋白與DNA的相互作用而促進(jìn)基因轉(zhuǎn)錄，然而組蛋白甲基化以殘基的修飾位點(diǎn)而決定基因的轉(zhuǎn)錄和抑制[43]。如同正常的豬受精胚胎，豬克隆胚胎的H3K27乙酰化水平從原核期到8-細(xì)胞期逐漸降低，這個(gè)時(shí)期對應(yīng)胚胎的基因組激活，但是在隨后的發(fā)育中H3K27乙?；惓44,45]。另外的研究顯示，豬克隆胚胎原核或2-細(xì)胞階段的H3K18乙酰化水平與隨后的發(fā)育能力呈正相關(guān)[46]。目前，已有很多研究利用組蛋白去乙?；种苿?histone deacetylase inhibitor, HDACi)調(diào)控豬克隆胚胎的組蛋白乙?；蕉鰪?qiáng)胚胎的發(fā)育能力，如Trichostatin、Scriptaid、oxamflatin、MGCD0103、丁酸鈉和丙戊酸等[30]。然而，這些方法并不能提高豬克隆胚胎的體內(nèi)發(fā)育效率。在豬中，1-細(xì)胞到4-細(xì)胞階段克隆胚胎的H3K9me2、H3K9me3和H4K20me3的表達(dá)水平異常高于體外受精胚胎，表明H3K9me2、H3K9me3和H4K20me3都有可能是豬克隆胚胎發(fā)育的表觀障礙[47]。利用組蛋白甲基轉(zhuǎn)移酶抑制劑BIX-01294可以顯著降低H3K9me2水平并提高了胚胎的體內(nèi)和體外發(fā)育效率[48]。H3K27me3也被發(fā)現(xiàn)是豬克隆胚胎發(fā)育的重要表觀障礙，降低H3K27me3的水平能提高胚胎的發(fā)育效率[49]。然而，早期胚胎階段改變組蛋白修飾并不能保證胚胎體內(nèi)的長期發(fā)育。最近研究表明，附植前胚胎中H3K27me3的印記丟失可能是胚胎附植后發(fā)育缺陷的主要原因[50]。

此外，X染色體失活異常也是克隆胚胎中的重要表觀重編程壁壘。X染色體失活是一種雌性特異的劑量補(bǔ)償機(jī)制，由X染色體連鎖的父源等位基因非編碼RNA調(diào)控完成。在發(fā)育異常的克隆動物中，基因異?；罨赡軙斐膳咛ブ滤阑蛘吡鳟a(chǎn)[51]，通過敲除或敲低能夠?qū)⑿∈蟮目寺⌒侍岣?~12倍[52,53]最近研究發(fā)現(xiàn)，妊娠30天和35天的異?？寺√旱幕虍惓８弑磉_(dá)，通過敲除供體細(xì)胞的基因能將豬的克隆效率提高6.9倍，表明表達(dá)失調(diào)與克隆胎兒發(fā)育異常有關(guān)[16]。

2.2 胎盤發(fā)育缺陷

胎盤作為連接母體與胎兒的重要橋梁，對胎兒的生長和發(fā)育具有重要調(diào)控作用。胎盤發(fā)育缺陷往往與多種妊娠并發(fā)癥相關(guān)，如先兆流產(chǎn)、IUGR、妊娠糖尿病和高血壓等，是導(dǎo)致胎兒發(fā)育不良甚至死亡的重要原因。豬胎盤屬于上皮絨毛膜胎盤，胚胎需要通過絨毛膜形成褶皺和內(nèi)陷加大與子宮內(nèi)膜的接觸面積才能從母體循環(huán)攝取充足的營養(yǎng)物質(zhì)[54]。對克隆豬的相關(guān)研究發(fā)現(xiàn)，相對于同期的人工授精胚胎，早期豬克隆胚胎的胚外組織形態(tài)異常，妊娠中期和足月胎盤的褶皺、滋養(yǎng)層及血管化發(fā)育不良[5,23,26,55,56]；胎盤中調(diào)控細(xì)胞凋亡、氧化應(yīng)激、血管形成、細(xì)胞增殖等過程的重要基因表達(dá)及信號通路異常，這些都可能是克隆豬宮內(nèi)發(fā)育不良的重要原因[5,57]。另外，最近研究發(fā)現(xiàn)，克隆豬宮內(nèi)發(fā)育遲緩或發(fā)育不良很可能與胎盤皺褶發(fā)育缺陷、膽汁酸轉(zhuǎn)運(yùn)和類固醇激素合成相關(guān)基因表達(dá)異常及脂肪酸轉(zhuǎn)運(yùn)蛋白4表達(dá)下調(diào)有關(guān)[27,56]。此外，臍帶異常影響血流而抑制胎兒的生長。Ao等[5]研究數(shù)據(jù)顯示，32.6% (15/46)存活的新生克隆豬的臍帶畸形，表現(xiàn)為臍帶膨大和閉塞性血栓，這些仔豬在出生后4天內(nèi)基本死亡，因?yàn)槟殠Щ我种蒲艿氖湛s和降低血流，增加胎兒發(fā)育異常的風(fēng)險(xiǎn)?？寺∝i的臍帶發(fā)育異?？赡芘c血管形成相關(guān)基因、、和的表達(dá)下調(diào)，以及參與抗氧化應(yīng)激和調(diào)控糖酵解的蛋白表達(dá)水平下調(diào)及細(xì)胞凋亡相關(guān)蛋白表達(dá)上調(diào)密切相關(guān)[8]。目前，胎盤發(fā)育缺陷仍然是SCNT發(fā)展中的一個(gè)主要障礙。因?yàn)榧词雇ㄟ^敲除和過表達(dá)將小鼠的克隆效率提高到20%，但是存活胚胎中仍然有胎盤異常的個(gè)體[50]。

3 提高豬克隆效率的有效方法

3.1 尋找豬克隆胚胎發(fā)育失敗的關(guān)鍵因子

供體細(xì)胞是生產(chǎn)克隆后代的遺傳基礎(chǔ)，供體核能否充分開啟轉(zhuǎn)錄組重編程將很大程度決定SCNT胚胎的發(fā)育命運(yùn)[58]。同濟(jì)大學(xué)高紹榮課題組結(jié)合胚胎活檢與單細(xì)胞測序方法對早期克隆胚胎進(jìn)行了詳細(xì)的轉(zhuǎn)錄組分析，發(fā)現(xiàn)H3K9me3去甲基化酶基因和分別在2-細(xì)胞和4-細(xì)胞期發(fā)育停滯的克隆胚胎中未被激活，過表達(dá)和能夠恢復(fù)這兩個(gè)發(fā)育階段的轉(zhuǎn)錄譜而顯著提高小鼠囊胚率(>95%)及出生率[59]。早期克隆胚胎的發(fā)育相關(guān)基因的正常表達(dá)與供體細(xì)胞的再甲基化密切相關(guān)。近期，高紹榮團(tuán)隊(duì)通過繪制小鼠附植前克隆胚胎的全基因組DNA甲基化圖譜，發(fā)現(xiàn)SCNT胚胎大范圍的DNA區(qū)域存在異常的DNA再甲基化，這種異常成為SCNT胚胎中合子基因和部分逆轉(zhuǎn)座子未能完全激活的關(guān)鍵障礙，通過抑制DNA甲基化酶和過表達(dá)組蛋白去甲基化酶都能顯著提高克隆小鼠的出生率[36]。供體細(xì)胞的組蛋白修飾模式未能重編程到受精卵狀態(tài)也會導(dǎo)致克隆胚胎發(fā)育失敗。美國哈佛大學(xué)張毅課題組結(jié)合轉(zhuǎn)錄組測序和染色質(zhì)免疫共沉淀數(shù)據(jù)分析基因組不同區(qū)域的組蛋白修飾與基因表達(dá)的聯(lián)系，發(fā)現(xiàn)供體細(xì)胞中H3K9me3是小鼠克隆胚胎發(fā)育的主要障礙，通過過表達(dá)或敲除H3K9me3甲基化酶基因以降低供體細(xì)胞的H3K9me3水平可將克隆效率提高8倍左右[60]。最近的研究顯示，過表達(dá)同樣也可以顯著提高克隆小鼠的出生率[61]。每個(gè)物種調(diào)控H3K9me3的模式不同，所以在人()[62]和牛()[63]中，分別過表達(dá)和才能顯著提高克隆胚胎的發(fā)育能力。在豬中，通過過表達(dá)能顯著下調(diào)克隆胚胎的H3K9me3水平而提高體外發(fā)育效率，但由于的啟動子區(qū)域富含H3K9me3，所以過表達(dá)不能支持克隆胚胎的長期發(fā)育[13]此外，過表達(dá)沒有改變克隆胚胎的H3K9me3水平和體外發(fā)育效率[64]。由此可見，需要深入解析早期豬克隆胚胎的表觀重編程變化才能有助于尋找豬克隆胚胎發(fā)育失敗的關(guān)鍵因子。

3.2 提高卵母細(xì)胞的成熟質(zhì)量

供體核在卵母細(xì)胞胞質(zhì)被誘導(dǎo)激活，所以卵母細(xì)胞很大程度上決定了重構(gòu)胚的發(fā)育能力。研究發(fā)現(xiàn)，體內(nèi)成熟卵母細(xì)胞作為核移植受體構(gòu)建的克隆胚胎的囊胚率和出生率均顯著高于體外成熟卵母細(xì)胞[65]，且經(jīng)產(chǎn)母豬來源的卵母細(xì)胞所獲得的克隆胚胎體外發(fā)育效率明顯高于后備母豬來源的卵母細(xì)胞[66]，表明卵母細(xì)胞的成熟質(zhì)量是影響克隆胚胎的發(fā)育能力的重要因素。卵母細(xì)胞成熟指第一次減數(shù)分裂前期到第二次減數(shù)分裂中期的過程，主要體現(xiàn)在核成熟和胞質(zhì)成熟，通常以第一極體排出作為核成熟的標(biāo)志，胞質(zhì)成熟表明卵母細(xì)胞具備受精能力和受精后的發(fā)育能力以及所需的物質(zhì)和能量儲備[67]。體內(nèi)成熟的卵母細(xì)胞在卵泡環(huán)境中能夠?qū)崿F(xiàn)細(xì)胞核和胞質(zhì)同步成熟，但是體外成熟的卵母細(xì)胞在體外培養(yǎng)體系中不能保證胞質(zhì)與細(xì)胞核的同步成熟，胞質(zhì)的不完全成熟是造成體外成熟卵母細(xì)胞發(fā)育能力低的重要原因[68]。轉(zhuǎn)錄組比較分析發(fā)現(xiàn)體內(nèi)與體外成熟卵母細(xì)胞中參與轉(zhuǎn)錄、細(xì)胞周期、轉(zhuǎn)運(yùn)和細(xì)胞蛋白代謝等生物學(xué)過程的基因表達(dá)具有明顯差異[69]。最近的一項(xiàng)研究已經(jīng)表明，改善卵母細(xì)胞發(fā)育質(zhì)量能顯著提高克隆胚胎的體內(nèi)發(fā)育效率，研究人員通過在卵母細(xì)胞體外成熟的培養(yǎng)基添加成纖維細(xì)胞生長因子2、白血病抑制因子和胰島素樣生長因子1顯著提高了克隆豬的囊胚率和出生率，窩均產(chǎn)仔數(shù)達(dá)到9頭左右，可能由于這些細(xì)胞因子使卵丘細(xì)胞具有不同的MAPK激活模式、增加卵丘細(xì)胞擴(kuò)張以及加快卵母細(xì)胞和卵丘細(xì)胞之間胞漿突起物的分離，進(jìn)而提高了卵母細(xì)胞的發(fā)育質(zhì)量[70]。卵泡液為卵母細(xì)胞的生長和成熟提供了適宜的微環(huán)境，在體外培養(yǎng)基中添加卵泡液也成為提升卵母細(xì)胞成熟質(zhì)量的重要途徑。Zhao等[71]發(fā)現(xiàn)卵母細(xì)胞體外培養(yǎng)基添加體內(nèi)成熟來源的卵泡液的克隆胚胎發(fā)育效率顯著高于未成熟卵泡液，可能由于體內(nèi)成熟卵泡液能提供更多促進(jìn)卵母細(xì)胞成熟的蛋白質(zhì)。卵母細(xì)胞在體外培養(yǎng)環(huán)境缺乏對自由基的清除能力，造成氧化應(yīng)激水平高而降低卵母細(xì)胞質(zhì)量，在體外培養(yǎng)基中添加自由基清除劑能夠一定程度提高卵母細(xì)胞質(zhì)量和重編程能力。研究發(fā)現(xiàn)，褪黑素通過降低卵母細(xì)胞的氧化應(yīng)激水平提高了SCNT及IVF胚胎的發(fā)育效率[72,73]。這些結(jié)果表明優(yōu)化體外培養(yǎng)體系來提高卵母細(xì)胞成熟質(zhì)量對于增強(qiáng)克隆胚胎的體內(nèi)發(fā)育能力具有重要作用。

3.3 降低代孕母豬的流產(chǎn)率

在克隆豬生產(chǎn)中，代孕母豬的懷孕率僅為50%左右，妊娠失敗的主要原因是克隆胎兒流產(chǎn)，且主要發(fā)生在妊娠第30~60天[17]?？寺√毫鳟a(chǎn)的主要原因是供體細(xì)胞的重編程錯(cuò)誤。Zhang等[38]發(fā)現(xiàn)克隆豬胎兒流產(chǎn)可能與胎兒和胎盤中印跡基因的表達(dá)異常及基因組重復(fù)區(qū)域的高甲基化有關(guān)。此外，品種、胎次、移植胚胎數(shù)量和排卵時(shí)間都會影響代孕母豬的懷孕率。與供體細(xì)胞品種相同的代孕母豬可獲得更高的分娩率，胚胎與受體品種間的差異會增加流產(chǎn)的風(fēng)險(xiǎn)[74]。雙側(cè)輸卵管移植的代孕母豬比單側(cè)移植的妊娠率和產(chǎn)仔率有顯著提高[75]。排卵前24 h進(jìn)行胚胎移植的母豬懷孕率和克隆效率都顯著高于排卵前6 h[76]。最近，Yu等[77]對誘導(dǎo)多能干細(xì)胞(Induced pluripotent stem cells, iPS)和成纖維細(xì)胞來源的克隆豬胎兒和胎盤進(jìn)行全基因組DNA甲基化和轉(zhuǎn)錄組測序分析，結(jié)果發(fā)現(xiàn)iPS克隆胎兒和胎盤的父本印跡基因處于異常的高甲基化狀態(tài)，超表達(dá)能降低iPS胎兒流產(chǎn)率而顯著提高iPS細(xì)胞的克隆效率，這些結(jié)果揭示的表達(dá)沉默很可能是克隆胚胎的流產(chǎn)主要原因。此外，日糧中添加營養(yǎng)物質(zhì)對于降低代孕母豬流產(chǎn)率具有一定作用，本課題組前期嘗試在妊娠第12~70天代孕母豬日糧中補(bǔ)充精氨酸，結(jié)果表明這種方式可以顯著提高代孕母豬的懷孕率(62.9%44.5%)[78]。因此，提高克隆胚胎的發(fā)育質(zhì)量、選擇適合的克隆胚胎移植方式以及調(diào)控克隆代孕母豬的營養(yǎng)水平都是降低代孕母豬流產(chǎn)率的重要途徑。

4 結(jié)語與展望

克隆豬的宮內(nèi)發(fā)育異常體現(xiàn)在胎兒和胎盤兩部分，核心是供體核表觀重編程，這里面涉及一系列復(fù)雜的表觀遺傳修飾變化。盡管供體核的表觀重編程錯(cuò)誤被認(rèn)為是克隆胚胎發(fā)育失敗的主要原因，但是其機(jī)制仍然不清楚。因此，需要系統(tǒng)和精細(xì)的分析重編程過程中染色體和表觀基因組的變化。

組蛋白修飾異常是克隆胚胎發(fā)育失敗的重要原因，因而解析蛋白質(zhì)與DNA的相互作用是一個(gè)重要的研究方向，其中染色質(zhì)可接近性與基因表達(dá)調(diào)控密切相關(guān)。目前，已有多種技術(shù)研究人和小鼠附植前胚胎染色質(zhì)可接近性而揭示胚胎發(fā)育過程中開放染色質(zhì)的調(diào)控規(guī)律，包括低通量脫氧核糖核酸酶I超敏感位點(diǎn)測序(low-input deoxyribonuclease I hy-persensitive site sequencing, liDNase-seq)技術(shù)[79,80]和轉(zhuǎn)座酶探究可接近性染色質(zhì)高通量測序(assay for transposase-accessible chromatin with high-throug-hput sequencing, ATAC-seq)技術(shù)[81,82]。此外，基于高通量測序的染色質(zhì)構(gòu)象捕獲(high-throughput/resolu-tion chromosome conformation capture, Hi-C)技術(shù)可以在全基因組范圍內(nèi)研究染色質(zhì)的空間構(gòu)象并揭示基因組的動態(tài)變化[83]。靶向調(diào)控供體細(xì)胞和克隆胚胎的表觀基因組是更加有效地提高克隆胚胎的發(fā)育質(zhì)量的一種途徑[84]。目前，很多研究研究已經(jīng)實(shí)現(xiàn)了基于CRISPR/Cas9的靶向表觀修飾。例如，Liu等[85]構(gòu)建了Tet1、Dnmt3a與失活的Cas9(dCas9)的融合蛋白可實(shí)現(xiàn)DNA甲基化的靶向編輯。CRISPR- dCas9-SunTag-p300core系統(tǒng)能夠靶向重塑多能基因的啟動子和增強(qiáng)子，并且同時(shí)調(diào)控多個(gè)基因的表達(dá)[86]。這些技術(shù)方法的應(yīng)用有助于研究人員解析供體核的表觀重編程過程，并解析豬克隆胚胎宮內(nèi)發(fā)育異常的分子機(jī)制。今后豬SCNT技術(shù)更應(yīng)注重克隆胚胎的體內(nèi)發(fā)育，提高出生率和健康率才能更好地發(fā)揮克隆豬的應(yīng)用價(jià)值。

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Progress on abnormal development of cloned pigs generated by somatic cell transfer nuclear

Zheng Ao1, Xiang Chen1, Zhenfang Wu2, Zicong Li2

Cloning, also known as somatic cell nuclear transfer (SCNT), is an asexual reproduction technique that reprograms differentiated cells to the totipotent state, and generates offspring with a genotype identical to the donor cells. Pig cloning technique holds great promise for propagating excellent breeding boars, generating genetically modified pigs, protecting rare and endangered pigs and studying the mechanisms of somatic cell nucleus reprogramming. However, cloned pigs suffer from various developmental defects, including low birth rate, low birth weight, and high stillbirth occurrence, neonatal mortality and congenital malformations, which severely hamper their applications. Errors in epigenetic reprog-ramming of donor nucleus are considered as the main causes of low cloning efficiency and abnormal embryonic develop-ment in cloned embryos and animals. However, most studies to correct the errors in epigenetic reprogramming of cloned pig embryos have not substantially improved the birth and survival rates of cloned pigs. In this review, we summarize the abnormal phenotypes, causes of abnormal development of cloned pigs and effective methods for improving pig cloning efficiency, thereby providing a reference for the future research to improve the development and survival rates of cloned pig embryos and cloned pigs.

SCNT; cloned pig; epigenetic reprogramming; abnormal development

2020-04-16;

2020-07-05

貴州大學(xué)引進(jìn)人才科研項(xiàng)目(編號：貴大人基合字(2019) 21號)資助[Supported by the Scientific Research Project of Guizhou University Talents Fund (No. GDRJHZ-2019-21)]

敖政，博士，講師，研究方向：動物遺傳育種與繁殖。E-mail: zheng780911@163.com

敖政。

10.16288/j.yczz.20-105

2020/7/30 10:01:58

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20200728.1634.002.html

(責(zé)任編委: 李明洲)