,1, 陈祥1, 吴珍芳2, 李紫聪2Progress on abnormal development of cloned pigs generated by somatic cell transfer nuclear
Zheng Ao,1, Xiang Chen1, Zhenfang Wu2, Zicong Li2通讯作者: 敖政。
编委: 李明洲
收稿日期:2020-04-16修回日期:2020-07-5网络出版日期:2020-10-20
| 基金资助: |
Received:2020-04-16Revised:2020-07-5Online:2020-10-20
| Fund supported: |
作者简介 About authors
敖政,博士,讲师,研究方向：动物遗传育种与繁殖。E-mail:
摘要
克隆又称体细胞核移植(somatic cell nuclear transfer, SCNT),是一种将已分化的细胞重编程恢复全能性而生产与供体细胞基因型完全相同后代的无性繁殖技术。猪的克隆技术具有重要的应用价值,包括扩繁优良种猪、制备基因修饰猪、保护珍贵和濒危猪种以及研究猪体细胞重编程机制。然而,克隆猪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问题,这些都严重影响了克隆猪的应用前景。供体核的表观重编程错误被认为是克隆效率低和胚胎发育异常的主要原因,但是目前大多数研究通过修正表观重编程错误并没有大幅度提高克隆猪的出生率和健康率。本文综述了克隆猪的异常表型、发育异常的原因以及提高猪克隆效率的有效方法,以期为提高克隆猪的成活率提供参考。
关键词：
Abstract
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 reprogramming of donor nucleus are considered as the main causes of low cloning efficiency and abnormal embryonic development 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.
Keywords：
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本文引用格式
敖政, 陈祥, 吴珍芳, 李紫聪. 体细胞克隆猪发育异常研究进展. 遗传[J], 2020, 42(10): 993-1003 doi:10.16288/j.yczz.20-105
Zheng Ao.
体细胞核移植(somatic cell transfer nuclear, SCNT)是将已分化的细胞重编程恢复全能型而生产与供体细胞基因型完全相同后代的一种无性繁殖技术[1]。从1996年克隆羊“多莉”诞生至今,多种哺乳动物的体细胞克隆都相继取得成功,特别是2018年初,中国科学家宣布成功获得了存活的体细胞克隆猴(Macaca fascicularis),这是体细胞克隆史上的一次重大突破[2]。猪(Sus scrofa) SCNT技术在农业、生物医学和基础研究领域具有重要的应用价值,包括扩繁优良种猪、制备基因修饰猪、保护珍贵和濒危猪种以及研究猪体细胞重编程机制[3,4]。然而,如表1所示,克隆猪的出生率低(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%)和初生重低等问题[5,6,7,8,9,10,11,12,13,14,15,16,17,18,19],这些都严重影响了克隆猪的应用前景。供体核的表观重编程错误被认为是克隆效率低和胚胎发育异常的主要原因,但是目前大多数研究通过修正表观重编程错误并没有大幅度提高克隆猪的出生率和健康率。因此,本文综述了克隆猪的异常表型、发育异常的原因以及提高猪克隆效率的有效方法,以期为提高克隆猪的成活率提供参考。
1 克隆猪发育异常
1.1 异常表型
Schmidt等[18]对815头克隆及转基因猪从出生至断奶进行了跟踪记录,结果发现新生克隆猪的死亡率为48%,畸形率为29.5%,尸检结果显示死亡克隆猪的主要生理缺陷分布在消化系统、循环系统、繁殖系统和骨骼肌系统,且存在诸多未发现的细微生理缺陷,这些生理缺陷严重影响机体正常的生理活动,是造成新生克隆猪死亡的直接或间接原因。因此,目前很难确定克隆猪死亡的具体机制,可能是一种或多种缺陷引发的结果。此外,很多新生克隆猪是无征兆的突然死亡,早期研究发现脑膜炎和血运障碍可能是仔猪突然死亡的主要原因[6]。克隆仔猪因屈肌腱和肘关节骨骼发育不良而导致仔猪站立失败,且新生克隆猪会表现出不佳的哺乳反射,因此需要人工及时喂养充足的初乳以增加仔猪的存活率[6]。克隆新生儿呼吸窘迫是克隆动物围产期死亡的原因之一,其病理特征包括不完整的肺扩张、肺泡塌陷、肺泡壁增厚、肺内表面活性剂稳态紊乱和透明膜异常,表明克隆新生儿的肺泡功能不全而造成呼吸窘迫[20,21]。克隆猪具有如肺动脉移位、肺畸形和肺发育不全等先天缺陷,这些都可能是仔猪围产期死亡的原因[18]。Park等[22]比较分析了出生后死亡及存活一个月克隆猪与同龄人工授精(artificial insemination, AI)猪肺脏的基因表达模式,在出生后死亡克隆猪中鉴定出121个差异表达基因(differentially expressed genes, DEGs),这些DEGs可能与克隆猪肺表面活性剂稳态失调和糖尿病性肾病等病理症状密切相关;在存活克隆猪中鉴定到154个DEGs,基因功能富集分析发现这些DEGs与肺泡发育延迟和MAPK信号通路下调有关,表明存活克隆猪仍然有器官功能异常的风险[23]。此外,部分克隆猪患有巨舌,这严重影响到仔猪的摄乳和呼吸,先天和环境因素导致这类仔猪几乎不能存活[6,18]。最近,本课题组研究发现,嘌呤代谢异常可能是克隆猪肾脏发生病理变化以及新生期死亡的重要原因[24]。
1.2 宫内发育不良
克隆胚胎的形态和功能异常是克隆猪出生率低的重要原因。猪胚胎在妊娠第10~12天之间经历从球状到管状和丝状的快速转变[25],Isom等[26]发现11.3%的猪克隆胚胎在妊娠第14天仍然是球状,表明部分克隆猪胚胎由于形态转变失败而停止发育。克隆胚胎的胚盘(embryonic disc, ED)和滋养层(trophectoderm, TE)与正常受精胚胎进行转录组比较,发现克隆胚胎的ED和TE中都有大量DEGs,其中ED的DEGs主要参与基因表达的表观遗传控制及microRNA介导的基因沉默和细胞凋亡,TE的DEGs主要与异常的代谢/异化途径和亚细胞的组织缺陷有关[26]。因此,即使胚胎能够附植成功,但大部分克隆胎儿和胎盘的发育仍然存在缺陷。Ruan等[16]在对妊娠30天和35天猪克隆胚胎进行转录组研究中发现,与AI胚胎相比,异常克隆胚胎中表达下调的基因数量比正常克隆胚胎更多,且大多数与胚胎发育相关的基因在异常克隆胚胎中未能激活表达。此外,大多数克隆猪具有宫内发育迟缓(intrauterine growth retardation, IUGR)特征,妊娠65天的克隆胎猪体重以及足月初生重显著低于AI猪[5,19,27]。初生重是一个重要的新生儿发病预测指标,克隆猪的初生重低可能是其出生后高频率死亡的重要原因[5]。Table 1
表1
表1代孕母猪怀孕率及克隆效率
Table 1
| 代孕母猪数 | 移植胚胎数 | 怀孕率(%) | 出生总仔数 | 活仔数 | 克隆效率(%) | 参考文献 |
|---|---|---|---|---|---|---|
| 24 | 5288 | 54.2 | 58 | 46 | 1.10 | [5] |
| 66 | 11,911 | 21.2 | 40 | — | 0.34 | [6] |
| 8 | 904 | 75 | 19 | 15 | 2.10 | [7] |
| 265 | 62,090 | 21.5 | 65 | 51 | 0.10 | [8] |
| 11 | 2884 | 72.7 | 12 | 10 | 0.42 | [9] |
| 193 | 18,649 | 56.5 | 318 | 243 | 1.71 | [10] |
| 328 | 92,005 | 57.6 | 488 | — | 0.53 | [11] |
| 4 | 1342 | 75 | 15 | 5 | 1.12 | [12] |
| 656 | 228,230 | 62 | 1070 | 810 | 0.47 | [13] |
| 79 | 13,620 | 63.3 | 177 | 119 | 1.30 | [14] |
| 4 | 1187 | 50 | 6 | 6 | 0.51 | [15] |
| 5 | 530 | 100 | 11 | — | 2.07 | [16] |
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2 克隆猪发育异常的原因
2.1 表观重编程错误
目前,供体核的表观重编程错误成为克隆胚胎发育异常的主流观点,主要包括DNA甲基化、组蛋白修饰和X染色体失活异常[28]。很多研究团队已经尝试通过修正这些表观遗传修饰错误来改变克隆胚胎的命运。DNA甲基化重塑是早期胚胎发育的关键步骤,涉及DNA去甲基化和再甲基化,由DNA甲基转移酶(DNA methyltransferase, DNMT)的催化作用完成[29]。供体细胞是克隆胚胎发育的起点,其表观修饰状态会直接影响克隆胚胎的发育能力[30]。研究发现,骨髓间充质干细胞(bone marrow stroma cell, BMSC)的囊胚率显著高于胎儿成纤维细胞(fetal fibroblasts, FF),可能由于BMSC来源的胚胎具有更低的Nanog和Pou5f1基因启动子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,不同类型的供体细胞的克隆胚胎发育效率具有明显差异,这很可能与供体细胞的分化程度有关[11,32]。DNA甲基化重塑异常几乎是所有克隆动物共有的特征,包括去甲基化不完全和再甲基化异常[33,34]。附植前胚胎的DNA甲基化需要经历特定的变化,其中早期和后期囊胚具有相似的DNA甲基化水平,且后期囊胚内细胞团(inner cell mass, ICM)的DNA甲基化水平显著高于TE[34]。然而,早期克隆囊胚的DNA甲基化水平显著高于后期囊胚,且后期囊胚的ICM与TE的DNA甲基化水平没有差异,表明DNA甲基化印记的异常变化可能造成胎儿和胎盘的发育缺陷[35]。最近,Gao等[36]研究发现附植前克隆胚胎经历异常DNA再甲基化,通过特定的DNMT抑制剂使DNA甲基化恢复到正常水平显著提高了小鼠(Mus musculus)的克隆效率,表明异常的DNA再甲基化也是抑制克隆胚胎发育的重要原因。DNA甲基化异常可能与Dnmt基因表达失调有关,抑制Dnmt基因的表达降低克隆胚胎中异常的高甲基化水平而利于基因的转录激活。研究发现,DNMT抑制剂RG108能促进DNA的主动和被动去甲基化以及Nanog转录而增强附植前克隆猪胚胎的发育能力[29,37]。对妊娠中期克隆胎儿流产的研究发现,胎儿的DNA重复区域PRE-1和卫星序列都呈现高度甲基化,且胎儿和胎盘中很多印迹基因表达异常以及H19的DMR3处于低甲基化水平,表明胎儿和胎盘发育异常都可能造成流产[38]。此外,DNA甲基化变化可能与克隆仔猪的异常表型有关。出生后的异常克隆猪与正常受精猪在全基因组的基因表达模式和DNA甲基化水平存在明显差异[39],且异常克隆猪的全基因组相对正常克隆猪更多是呈现低DNA甲基化水平[40]。然而,不能定论异常克隆仔猪的全基因组都是处于低DNA甲基化水平,因为其CpG岛区域具有更高的DNA甲基化水平[40]。尽管表型正常的克隆猪与普通猪的基因表达模式高度相似,但是克隆猪呈现更多不同的单拷贝序列DNA甲基化模式,DNA甲基化水平的差异可能会影响克隆猪的组织或器官发育[41]。哺乳动物基因组的组蛋白的N末端有很多修饰形式,包括甲基化、乙酰化、磷酸化和泛素化等[42]。这些修饰可以影响组蛋白与染色质的相互作用而调控基因的转录[28]。常见的组蛋白修饰是甲基化和乙酰化。组蛋白乙酰化可以减弱组蛋白与DNA的相互作用而促进基因转录,然而组蛋白甲基化以残基的修饰位点而决定基因的转录和抑制[43]。如同正常的猪受精胚胎,猪克隆胚胎的H3K27乙酰化水平从原核期到8-细胞期逐渐降低,这个时期对应胚胎的基因组激活,但是在随后的发育中H3K27乙酰化异常[44,45]。另外的研究显示,猪克隆胚胎原核或2-细胞阶段的H3K18乙酰化水平与随后的发育能力呈正相关[46]。目前,已有很多研究利用组蛋白去乙酰化抑制剂(histone deacetylase inhibitor, HDACi)调控猪克隆胚胎的组蛋白乙酰化水平而增强胚胎的发育能力,如Trichostatin、Scriptaid、oxamflatin、MGCD0103、丁酸钠和丙戊酸等[30]。然而,这些方法并不能提高猪克隆胚胎的体内发育效率。在猪中,1-细胞到4-细胞阶段克隆胚胎的H3K9me2、H3K9me3和H4K20me3的表达水平异常高于体外受精胚胎,表明H3K9me2、H3K9me3和H4K20me3都有可能是猪克隆胚胎发育的表观障碍[47]。利用组蛋白甲基转移酶抑制剂BIX-01294可以显著降低H3K9me2水平并提高了胚胎的体内和体外发育效率[48]。H3K27me3也被发现是猪克隆胚胎发育的重要表观障碍,降低H3K27me3的水平能提高胚胎的发育效率[49]。然而,早期胚胎阶段改变组蛋白修饰并不能保证胚胎体内的长期发育。最近研究表明,附植前胚胎中H3K27me3的印记丢失可能是胚胎附植后发育缺陷的主要原因[50]。
此外,X染色体失活异常也是克隆胚胎中的重要表观重编程壁垒。X染色体失活是一种雌性特异的剂量补偿机制,由X染色体连锁的父源等位基因非编码RNA Xist调控完成。在发育异常的克隆动物中,Xist基因异常活化可能会造成胚胎致死或者流产[51],通过敲除或敲低Xist能够将小鼠的克隆效率提高8~12倍[52,53]。最近研究发现,妊娠30天和35天的异常克隆胎儿的Xist基因异常高表达,通过敲除供体细胞的Xist基因能将猪的克隆效率提高6.9倍,表明Xist表达失调与克隆胎儿发育异常有关[16]。
2.2 胎盘发育缺陷
胎盘作为连接母体与胎儿的重要桥梁,对胎儿的生长和发育具有重要调控作用。胎盘发育缺陷往往与多种妊娠并发症相关,如先兆流产、IUGR、妊娠糖尿病和高血压等,是导致胎儿发育不良甚至死亡的重要原因。猪胎盘属于上皮绒毛膜胎盘,胚胎需要通过绒毛膜形成褶皱和内陷加大与子宫内膜的接触面积才能从母体循环摄取充足的营养物质[54]。对克隆猪的相关研究发现,相对于同期的人工授精胚胎,早期猪克隆胚胎的胚外组织形态异常,妊娠中期和足月胎盘的褶皱、滋养层及血管化发育不良[5,23,26,55,56];胎盘中调控细胞凋亡、氧化应激、血管形成、细胞增殖等过程的重要基因表达及信号通路异常,这些都可能是克隆猪宫内发育不良的重要原因[5,57]。另外,最近研究发现,克隆猪宫内发育迟缓或发育不良很可能与胎盘皱褶发育缺陷、胆汁酸转运和类固醇激素合成相关基因表达异常及脂肪酸转运蛋白4表达下调有关[27,56]。此外,脐带异常影响血流而抑制胎儿的生长。Ao等[5]研究数据显示,32.6% (15/46)存活的新生克隆猪的脐带畸形,表现为脐带膨大和闭塞性血栓,这些仔猪在出生后4天内基本死亡,因为脐带畸形抑制血管的收缩和降低血流,增加胎儿发育异常的风险。克隆猪的脐带发育异常可能与血管形成相关基因Vegf、Vegfr1、Ang1和Ang2的表达下调,以及参与抗氧化应激和调控糖酵解的蛋白表达水平下调及细胞凋亡相关蛋白表达上调密切相关[8]。目前,胎盘发育缺陷仍然是SCNT发展中的一个主要障碍。因为即使通过敲除Xist和过表达Kdm4a将小鼠的克隆效率提高到20%,但是存活胚胎中仍然有胎盘异常的个体[50]。3 提高猪克隆效率的有效方法
3.1 寻找猪克隆胚胎发育失败的关键因子
供体细胞是生产克隆后代的遗传基础,供体核能否充分开启转录组重编程将很大程度决定SCNT胚胎的发育命运[58]。同济大学高绍荣课题组结合胚胎活检与单细胞测序方法对早期克隆胚胎进行了详细的转录组分析,发现H3K9me3去甲基化酶基因Kdm4b和Kdm5b分别在2-细胞和4-细胞期发育停滞的克隆胚胎中未被激活,过表达Kdm4b和Kdm5b能够恢复这两个发育阶段的转录谱而显著提高小鼠囊胚率(>95%)及出生率[59]。早期克隆胚胎的发育相关基因的正常表达与供体细胞的再甲基化密切相关。近期,高绍荣团队通过绘制小鼠附植前克隆胚胎的全基因组DNA甲基化图谱,发现SCNT胚胎大范围的DNA区域存在异常的DNA再甲基化,这种异常成为SCNT胚胎中合子基因和部分逆转座子未能完全激活的关键障碍,通过抑制DNA甲基化酶和过表达组蛋白去甲基化酶都能显著提高克隆小鼠的出生率[36]。供体细胞的组蛋白修饰模式未能重编程到受精卵状态也会导致克隆胚胎发育失败。美国哈佛大学张毅课题组结合转录组测序和染色质免疫共沉淀数据分析基因组不同区域的组蛋白修饰与基因表达的联系,发现供体细胞中H3K9me3是小鼠克隆胚胎发育的主要障碍,通过过表达Kdm4d或敲除H3K9me3甲基化酶基因Suv39h1/2以降低供体细胞的H3K9me3水平可将克隆效率提高8倍左右[60]。最近的研究显示,过表达Kdm6a同样也可以显著提高克隆小鼠的出生率[61]。每个物种调控H3K9me3的模式不同,所以在人(Homo sapiens)[62]和牛(Bos taurus)[63]中,分别过表达Kdm4a和Kdm4e才能显著提高克隆胚胎的发育能力。在猪中,通过过表达Kdm4a能显著下调克隆胚胎的H3K9me3水平而提高体外发育效率,但由于Xist的启动子区域富含H3K9me3,所以过表达Kdm4a不能支持克隆胚胎的长期发育[13]。此外,过表达Kdm4b/4d没有改变克隆胚胎的H3K9me3水平和体外发育效率[64]。由此可见,需要深入解析早期猪克隆胚胎的表观重编程变化才能有助于寻找猪克隆胚胎发育失败的关键因子。3.2 提高卵母细胞的成熟质量
供体核在卵母细胞胞质被诱导激活,所以卵母细胞很大程度上决定了重构胚的发育能力。研究发现,体内成熟卵母细胞作为核移植受体构建的克隆胚胎的囊胚率和出生率均显著高于体外成熟卵母细胞[65],且经产母猪来源的卵母细胞所获得的克隆胚胎体外发育效率明显高于后备母猪来源的卵母细胞[66],表明卵母细胞的成熟质量是影响克隆胚胎的发育能力的重要因素。卵母细胞成熟指第一次减数分裂前期到第二次减数分裂中期的过程,主要体现在核成熟和胞质成熟,通常以第一极体排出作为核成熟的标志,胞质成熟表明卵母细胞具备受精能力和受精后的发育能力以及所需的物质和能量储备[67]。体内成熟的卵母细胞在卵泡环境中能够实现细胞核和胞质同步成熟,但是体外成熟的卵母细胞在体外培养体系中不能保证胞质与细胞核的同步成熟,胞质的不完全成熟是造成体外成熟卵母细胞发育能力低的重要原因[68]。转录组比较分析发现体内与体外成熟卵母细胞中参与转录、细胞周期、转运和细胞蛋白代谢等生物学过程的基因表达具有明显差异[69]。最近的一项研究已经表明,改善卵母细胞发育质量能显著提高克隆胚胎的体内发育效率,研究人员通过在卵母细胞体外成熟的培养基添加成纤维细胞生长因子2、白血病抑制因子和胰岛素样生长因子1显著提高了克隆猪的囊胚率和出生率,窝均产仔数达到9头左右,可能由于这些细胞因子使卵丘细胞具有不同的MAPK激活模式、增加卵丘细胞扩张以及加快卵母细胞和卵丘细胞之间胞浆突起物的分离,进而提高了卵母细胞的发育质量[70]。卵泡液为卵母细胞的生长和成熟提供了适宜的微环境,在体外培养基中添加卵泡液也成为提升卵母细胞成熟质量的重要途径。Zhao等[71]发现卵母细胞体外培养基添加体内成熟来源的卵泡液的克隆胚胎发育效率显著高于未成熟卵泡液,可能由于体内成熟卵泡液能提供更多促进卵母细胞成熟的蛋白质。卵母细胞在体外培养环境缺乏对自由基的清除能力,造成氧化应激水平高而降低卵母细胞质量,在体外培养基中添加自由基清除剂能够一定程度提高卵母细胞质量和重编程能力。研究发现,褪黑素通过降低卵母细胞的氧化应激水平提高了SCNT及IVF胚胎的发育效率[72,73]。这些结果表明优化体外培养体系来提高卵母细胞成熟质量对于增强克隆胚胎的体内发育能力具有重要作用。3.3 降低代孕母猪的流产率
在克隆猪生产中,代孕母猪的怀孕率仅为50%左右,妊娠失败的主要原因是克隆胎儿流产,且主要发生在妊娠第30~60天[17]。克隆胎儿流产的主要原因是供体细胞的重编程错误。Zhang等[38]发现克隆猪胎儿流产可能与胎儿和胎盘中印迹基因的表达异常及基因组重复区域的高甲基化有关。此外,品种、胎次、移植胚胎数量和排卵时间都会影响代孕母猪的怀孕率。与供体细胞品种相同的代孕母猪可获得更高的分娩率,胚胎与受体品种间的差异会增加流产的风险[74]。双侧输卵管移植的代孕母猪比单侧移植的妊娠率和产仔率有显著提高[75]。排卵前24 h进行胚胎移植的母猪怀孕率和克隆效率都显著高于排卵前6 h[76]。最近,Yu等[77]对诱导多能干细胞(Induced pluripotent stem cells, iPS)和成纤维细胞来源的克隆猪胎儿和胎盘进行全基因组DNA甲基化和转录组测序分析,结果发现iPS克隆胎儿和胎盘的父本印迹基因Rtl1处于异常的高甲基化状态,超表达Rtl1能降低iPS胎儿流产率而显著提高iPS细胞的克隆效率,这些结果揭示Rtl1的表达沉默很可能是克隆胚胎的流产主要原因。此外,日粮中添加营养物质对于降低代孕母猪流产率具有一定作用,本课题组前期尝试在妊娠第12~70天代孕母猪日粮中补充精氨酸,结果表明这种方式可以显著提高代孕母猪的怀孕率(62.9% vs. 44.5%)[78]。因此,提高克隆胚胎的发育质量、选择适合的克隆胚胎移植方式以及调控克隆代孕母猪的营养水平都是降低代孕母猪流产率的重要途径。4 结语与展望
克隆猪的宫内发育异常体现在胎儿和胎盘两部分,核心是供体核表观重编程,这里面涉及一系列复杂的表观遗传修饰变化。尽管供体核的表观重编程错误被认为是克隆胚胎发育失败的主要原因,但是其机制仍然不清楚。因此,需要系统和精细的分析重编程过程中染色体和表观基因组的变化。组蛋白修饰异常是克隆胚胎发育失败的重要原因,因而解析蛋白质与DNA的相互作用是一个重要的研究方向,其中染色质可接近性与基因表达调控密切相关。目前,已有多种技术研究人和小鼠附植前胚胎染色质可接近性而揭示胚胎发育过程中开放染色质的调控规律,包括低通量脱氧核糖核酸酶I超敏感位点测序(low-input deoxyribonuclease I hypersensitive site sequencing, liDNase-seq)技术[79,80]和转座酶探究可接近性染色质高通量测序(assay for transposase-accessible chromatin with high-throughput sequencing, ATAC-seq)技术[81,82]。此外,基于高通量测序的染色质构象捕获(high-throughput/resolution chromosome conformation capture, Hi-C)技术可以在全基因组范围内研究染色质的空间构象并揭示基因组的动态变化[83]。靶向调控供体细胞和克隆胚胎的表观基因组是更加有效地提高克隆胚胎的发育质量的一种途径[84]。目前,很多研究研究已经实现了基于CRISPR/Cas9的靶向表观修饰。例如,Liu等[85]构建了Tet1、Dnmt3a与失活的Cas9(dCas9)的融合蛋白可实现DNA甲基化的靶向编辑。CRISPR- dCas9-SunTag-p300core系统能够靶向重塑多能基因Oct4的启动子和增强子,并且同时调控多个基因的表达[86]。这些技术方法的应用有助于研究人员解析供体核的表观重编程过程,并解析猪克隆胚胎宫内发育异常的分子机制。今后猪SCNT技术更应注重克隆胚胎的体内发育,提高出生率和健康率才能更好地发挥克隆猪的应用价值。
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被引期刊影响因子
DOI:10.1098/rstb.2011.0329URLPMID:23166393 [本文引用: 1]
Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.
[本文引用: 1]
[本文引用: 1]
DOI:10.3389/fgene.2018.00360URLPMID:30233645 [本文引用: 1]
Pigs serve as an important agricultural resource and animal model in biomedical studies. Efficient and precise modification of pig genome by using recently developed gene editing tools has significantly broadened the application of pig models in various research areas. The three types of site-specific nucleases, namely, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein, are the main gene editing tools that can efficiently introduce predetermined modifications, including knockouts and knockins, into the pig genome. These modifications can confer desired phenotypes to pigs to improve production traits, such as optimal meat production, enhanced feed digestibility, and disease resistance. Besides, given their genetic, anatomic, and physiologic similarities to humans, pigs can also be modified to model human diseases or to serve as an organ source for xenotransplantation to save human lives. To date, many genetically modified pig models with agricultural or biomedical values have been established by using gene editing tools. These pig models are expected to accelerate research progress in related fields and benefit humans.
URLPMID:28864025 [本文引用: 6]
DOI:10.1002/pmic.200401079URLPMID:15832370 [本文引用: 4]
In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, five piglets were stillborn, 22 scNT piglets died suddenly within the first week of life, and 1 piglet died after 40 days. Twelve scNT piglets are still healthy. The birth weights of compromised scNT piglets in comparison with those of normal scNT piglets are significantly reduced (0.80 +/- 0.29 vs 1.27 +/- 0.30 kg, p < 0.05), in spite of longer gestation (114 versus 120 day). Significant findings from histological examinations showed that approximately 25% (7/28) of scNT piglets showed severe congestion of lung and liver or neutrophilic inflammation in brain indicating that unexpected phenotypes can appear as a result of somatic cell cloning. Two-dimensional gel electrophoresis experiments revealed changes in the responses of several detoxification-related proteins related to stress and inflammation and found significant alterations in myocardium-specific proteins, indicating hemodynamic disorder. scNT piglets that survived to adulthood did not show any abnormality except skin and hair color depigmentation. The present study suggests that cerebromeningitis and hemodynamic disorder are a major risk factor for sudden early death of scNT piglets. Although we cannot completely exclude the possibility that scNT piglets are susceptible to specific respiratory infections, our data suggests that the early death of scNT clones is due to cardiopulmonary functional abnormalities and cerebromeningitis.
DOI:10.1262/jrd.20038URLPMID:18490858 [本文引用: 1]
The aim of the present study was to examine the production efficiency of cloned pigs by serial somatic cell nuclear transfer (SCNT) and to ascertain any changes in the telomere lengths of multiple generations of pigs. Using fetal fibroblasts as the starting nuclear donor cells, porcine salivary gland progenitor cells were collected from the resultant first-generation cloned pigs to successively produce second- and third-generation clones, with no significant differences in production efficiency, which ranged from 1.4% (2/140) to 3.3% (13/391) among the 3 generations. The average telomere lengths (terminal restriction fragment values) for the first, second and third generation clones were 16.3, 18.1 and 20.5 kb, respectively, and were comparable to those in age-matched controls. These findings suggest that third-generation cloned pigs can be produced by serial somatic cell cloning without compromising production efficiency and that the telomere lengths of cloned pigs from the first to third generations are normal.
DOI:10.1186/1471-2164-10-511URLPMID:19889237 [本文引用: 2]
BACKGROUND: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. RESULTS: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. CONCLUSION: These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development.
URLPMID:23469059 [本文引用: 1]
DOI:10.1186/1472-6750-13-43URLPMID:23688045 [本文引用: 1]
BACKGROUND: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. RESULTS: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. CONCLUSION: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
DOI:10.1089/cell.2012.0042URL [本文引用: 2]
Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born / total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P, D, LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P, D, L, Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 +/- 0.34) was similar with that associated with P, D, L, Y-FFBs (2.72 +/- 0.29), but was significantly higher than that resulted from P, D, L, Y-AFBs (1.47 +/- 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P, D, L, Y-AFBs and P, D, L, Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs.
DOI:10.1007/s13238-014-0034-3URLPMID:24627095 [本文引用: 1]
Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 mumol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
DOI:10.1089/cell.2015.0037URLPMID:26655078 [本文引用: 2]
Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the first report of large-scale analysis of porcine cell nuclear transfer that provides important data for potential industrialization of HMC technology.
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DOI:10.1007/s13205-016-0525-4URLPMID:28330290 [本文引用: 1]
Somatic cloning, also known as somatic cell nuclear transfer (SCNT), is a promising technology which has been expected to rapidly extend the population of elaborately selected breeding boars with superior production performance. Chinese Guike No. 1 pig breed is a novel swine specialized strain incorporated with the pedigree background of Duroc and Chinese Luchuan pig breeds, thus inherits an excellent production performance. The present study was conducted to establish somatic cloning procedures of adult breeding boars from the Chinese Guike No. 1 specialized strain. Ear skin fibroblasts were first isolated from a three-year-old Chinese Guike No. 1 breeding boar, and following that, used as donor cell to produce nuclear transfer embryos. Such cloned embryos showed full in vitro development and with the blastocyst formation rate of 18.4 % (37/201, three independent replicates). Finally, after transferring of 1187 nuclear transfer derived embryos to four surrogate recipients, six live piglets with normal health and development were produced. The overall cloning efficiency was 0.5 % and the clonal provenance of such SCNT derived piglets was confirmed by DNA microsatellite analysis. All of the cloned piglets were clinically healthy and had a normal weight at 1 month of age. Collectively, the first successful cloning of an adult Chinese Guike No. 1 breeding boar may lay the foundation for future improving the pig production industry.
DOI:10.1016/j.stemcr.2017.12.015URL [本文引用: 3]
DOI:10.1071/RD13329URL [本文引用: 2]
DOI:10.1016/j.theriogenology.2015.05.037URLPMID:26166169 [本文引用: 4]
Important factors contributing to the well-known high mortality of piglets produced by SCNT are gross malformations of vital organs. The aim of the present retrospective study was to describe malformations found in cloned piglets, transgenic or not, dying or culled before weaning on Day 28. Large White (LW) embryos were transferred to 78 LW recipients, while 72 recipients received Gottingen embryos (67 transgenic and five not transgenic) and 56 received Yucatan embryos (43 transgenic and 13 not transgenic). Overall pregnancy rate was 76%, and there were more abortions in recipients with minipig embryos than in those with LW embryos (26% and 24% vs. 6%). Piglets (n = 815) were born from 128 sows with 6.5 +/- 0.4 full-born piglets per litter. The overall rate of stillborn piglets was 21% of all born with the number of stillborn piglets ranging from one to nine in a litter. The mortality of the surviving piglets during the first month was 48%. Thus, altogether 58% of the full-born piglets died before weaning. In 87 of the 128 litters (68%), one to 12 of the piglets showed major or minor malformations. Malformations were found in 232 piglets (29.5% of all born). A single malformation was registered in 152 piglets, but several piglets showed two (n = 58) or more (n = 23) malformations (7.4% and 2.8% of all born, respectively). A significantly higher malformation rate was found in transgenic Gottingen and Yucatan piglets (32% and 46% of all born, respectively) than in nontransgenic LW (17%). There was a gender difference in the transgenic minipigs because male piglets had a higher rate of malformations (49.1%) than females (29.7%). The most common defects in the cloned piglets were in the digestive (12.2%), circulatory (9.4%), reproductive (11.3%), and musculoskeletal (9.1%) systems. Malformations of the musculoskeletal system were most frequent in Gottingen (16.3% vs. approximately 5.5% in the two other breeds), whereas abnormal cardiopulmonary systems were most frequent in Yucatan piglets (26.9% vs. 2.1% in LW and 5.3% in Gottingen). In conclusion, these results show that pig cloning results in a considerable loss of piglets and that many of these can be related to various malformations that all are also seen in noncloned piglets. Because approximately half of the cloned piglets still survive, even with eventual unknown minor malformations, use of pigs as models for human diseases is still realistic. However, continued efforts are needed to further reduce the level of malformations.
DOI:10.1089/clo.2006.0079URLPMID:17579555 [本文引用: 2]
While somatic cell nuclear transfer (SCNT) has been successful in several species, many pregnancies are lost and anomalies are found in fetal and perinatal stages. In this study SCNT and artificial inseminations (AI) populations were compared for litter size, average birth weight, piglets alive at birth, stillborn, mummies, dead at the first week, intrauterine growth restriction (IUGR) and large for gestational age (LGA). Twenty-three SCNT litters (143 individuals) were compared to 112 AI litters (1300 individuals). Litter size average was 11.5 for AI and 6.2 for SCNT. Litter weight and average birth weight adjusted by litter size were significantly (p < 0.05) higher in AI than in SCNT litters. The SCNT population had a significant (p < 0.01) increase in the number of IUGRs per litter with LSmeans 7.2 +/- 1.4 versus 19.4 +/- 3.5 and means 8.0 +/- 10.8 versus 15.5 +/- 24.5 for AI and SCNT, respectively. Additionally, there was a trend for higher postnatal mortality and stillbirths in the SCNT population. These findings demonstrate that there are some differences between SCNT-derived and AI litters. SCNT-derived pigs are excellent models to study epigenetic factors and genes involved in IUGRs, and to develop effective means to improve fetal growth in humans and animals.
DOI:10.1002/mrd.22956URLPMID:29388718 [本文引用: 1]
Neonatal respiratory distress is a major mortality factor in cloned animals, but the pathogenesis of this disease is rarely investigated. In this study, four neonatal cloned cattle, born after full-term gestation, exhibited symptoms of neonatal respiratory distress syndrome (NRDS), which included symptoms of hyaline membrane disease as well as disordered surfactant homeostasis in their collapsed lungs. No differences in DNA methylation or histone modifications correlated with the suppressed SPB and SPC transcription observed in the cloned cattle group (p > 0.05), whereas TTF-1 occupancy at SPB and SPC promoter regions in cloned cattle was significantly reduced to 24% and 20% that of normal lungs, respectively (SPB, p < 0.05; SPC, p < 0.01). Decreased TTF1 expression, dysregulation of SPB and SPC transcription by TTF-1, and disordered proteolytic processing of Surfactant protein B precursor together potentially contribute to the disruption of surfactant homeostasis and NRDS in bovine clones. Elucidation of the associated mechanisms should facilitate the development of novel preventive or therapeutic strategies to reduce the mortality rate of cloned animals and to improve the efficiency of SCNT technology.
DOI:10.1002/mrd.22836URLPMID:28513901 [本文引用: 1]
Respiratory distress is a major cause of mortality in cloned neonatal animals, but its pathogenesis remains poorly understood. Here, we used necropsy and histology procedures to evaluate the lungs of cloned neonatal bovines dying of respiratory distress, finding incomplete lung dilation, alveolar collapse, and thickened alveolar walls. Comparison of the transcriptomes between collapsed lungs of cloned bovines and their normal counterparts revealed 1373 differentially expressed genes in collapsed lungs (p < 0.05, fold change >1.5 or <1.5(-1) ), many of which were associated with surfactant biosynthesis, secretion, transport, recycling, and degradation. ERK/MAPK and Notch signaling pathways were among the canonical pathways relevant to surfactant homeostasis. Expression of the genes encoding Surfactant protein B (SPB) and Surfactant protein C (SPC)-which control surfactant lipid packing, spreading, and stability-were significantly lower in collapsed lungs of cloned neonates at the transcript (p < 0.01) and protein levels (p < 0.05) relative to that in normal lungs. Thus, our results provide an initial view into the changes in gene expression in cloned newborns with lung collapse and respiratory distress, and present a valuable resource for developing novel preventive or therapeutic strategies to reduce the mortality rate of cloned animals and to improve the efficiency of somatic cell nuclear transfer technology.
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DOI:10.1089/cell.2010.0088URL [本文引用: 2]
Although numerous mammalian species have been successfully cloned by somatic cell nuclear transfer (SCNT), little is known about gene expression of cloned pigs by SCNT. In the present study, expression profiles of 1-month-old cloned pigs generated from fetal fibroblasts (n = 5) were compared to those of age-matched controls (n = 5) using a 13K oligonucleotide microarray. The brain, kidney, and lung were chosen for microarray analysis to represent tissues from endoderm, mesoderm, and ectoderm in origin. In clones, 179 and 154 genes were differentially expressed in the kidney and the lung, respectively (fold change >2, p<0.05, false discovery rate = 0.05), whereas only seven genes were differentially expressed in the brain of clones. Functional analysis of the differentially expressed genes revealed that they were enriched in diabetic nephropathy in the kidney, delayed alveologenesis as well as downregulated MAPK signaling pathways in the lung, which was accompanied with collapsed alveoli in the histological examination of the lung. To evaluate whether the gene expression anomalies are associated with changes in DNA methylation, global concentration of the methylated cytosine was measured in lung DNA by HPLC. Clones were significantly hypermethylated (5.72%) compared to the controls (4.13%). Bisulfite-pyrosequencing analyses of the promoter regions of differentially expressed genes, MYC and Period 1 (PER1), however, did not show any differences in the degree of DNA methylation between controls and clones. Together, these findings demonstrate that cloned pigs have altered gene expression that may potentially cause organ dysfunction.
DOI:10.1530/REP-20-0143URLPMID:32413846 [本文引用: 1]
Cloned pigs generated by the somatic cell transfer nuclear (SCNT) technique are highly valuable for agriculture, biomedicine, and life sciences. However, the neonatal mortality rate of cloned pigs is very high. The reasons causing the massive loss of cloned pigs during their neonatal ages are unclear. In the present study, we found that the neonatal death of cloned pigs was associated with aberrant purine metabolism, impaired renal morphology and function, and decreased hepatic Hprt1 expression. The downregulation of Hprt1, a key purine metabolism regulation gene, in the liver was responsible for the elevation of an important purine metabolite, uric acid, in the serum, causing abnormalities in kidney morphology and function and leading to death of neonatal cloned pigs. This study provided insights into the pathophysiological mechanisms underlying the neonatal death of clone pigs, and results will help improve their survival rate.
DOI:10.1016/j.diff.2013.11.005URL [本文引用: 1]
The litter-bearing pig is an invaluable model for research in reproductive biology. Spherical pig blastocysts on Day 10 of pregnancy undergo rapid morphological changes to tubular and then filamentous forms by Day 12 and a filamentous. conceptus of almost 1 m in length by Day 16 of pregnancy. Thus, trophectoderm of each conceptus achieves intimate contact with luminal uterine epithelium (LE) for exchange of nutrients, gases, hormones, growth factors and other key molecules for survival and development. Estrogens secreted between Days 11 and 13 of pregnancy signals pregnancy recognition to ensure that nutrients and prostaglandin F2-alpha (PGF) are secreted into the uterine lumen (exocrine secretion) rather than into the uterine vein (endocrine secretion) which would lead to regression of the corpora lutea (CL) and failure to maintain pregnancy. Pigs have a true epitheliochorial placenta. The fluid filled amnion bouys the embryo so that it develops symmetrically. The allantois fills with allantoic fluid to expand contact of the chorioallantois with uterine LE, and the allanotois supports the vascular system of the placenta. The chorion/trophectoderm in direct contact with uterine LE exchanges gases and nutrients and forms unique structures call areolae that absorb nutrient-rich secretions from uterine glands and transports them directly into fetal blood. The period from Days 20 to 70 of pregnancy is for placental growth in preparation for rapid fetal growth between Days 70 and 114 (term) of gestation. Maturation of the fetal hypothalamic-pituitary-adrenal axis leads to increases in secretion of cortisol from the fetal adrenal glands. Cortisol sets in motion secretion of estrogens, oxytocin, relaxin and prolactin, as well as increases in their receptors, which are required for delivery of piglets and for initiation of lactation and expression of maternal behavior. This review provides details of gestation in the pig with respect to uterine biology, implantation, placentation, fetal development and parturition. (C) 2013 International Society of Differentiation. Published by Elsevier B.V.
DOI:10.1152/physiolgenomics.00094.2012URLPMID:23695885 [本文引用: 3]
Substantial mortality of in vitro manipulated porcine embryos is observed during peri-attachment development. Herein we describe our efforts to characterize the transcriptomes of embryonic disc (ED) and trophectoderm (TE) cells from porcine embryos derived from in vivo fertilization, in vitro fertilization (IVF), parthenogenetic oocyte activation (PA), and somatic cell nuclear transfer (SCNT) on days 10, 12, and 14 of gestation. The IVF, PA, and SCNT embryos were generated with in vitro matured oocytes and were cultured overnight in vitro before being transferred to recipient females. Sequencing of cDNA from the resulting embryonic samples was accomplished with the Genome Analyzer IIx platform from Illumina. Reads were aligned to a custom-built swine transcriptome. A generalized linear model was fit for ED and TE samples separately, accounting for embryo type, gestation day, and their interaction. Those genes with significant differences between embryo types were characterized in terms of gene ontologies and KEGG pathways. Transforming growth factor-beta signaling was downregulated in the EDs of IVF embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signaling were aberrantly regulated. Expression of genes involved in chromatin modification, gene silencing by RNA, and apoptosis was significantly disrupted in ED cells from SCNT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various embryo types during peri-attachment development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from assisted reproductive technologies.
DOI:10.1002/mrd.23102URLPMID:30618166 [本文引用: 2]
Piglets cloned by somatic cell nuclear transfer (SCNT) show a high incidence of malformations and a high death rate during the perinatal period. To investigate the underlying mechanisms for abnormal development of cloned pig fetuses, we compared body weight, amniotic fluid (AF) metabolome, and placental transcriptome between SCNT- and artificial insemination (AI)-derived pig fetuses. Results showed that the body weight of SCNT pig fetuses was significantly lower than that of AI pig fetuses. The identified differential metabolites between the two groups of AF were mainly involved in bile acids and steroid hormones. The levels of all detected bile acids in SCNT AF were significantly higher than those in AI AF. The increase in the AF bile acid levels in SCNT fetuses was linked with the downregulation of placental bile acid transporter expression and the abnormal development of placental folds (PFs), both of which negatively affected the transfer of bile acids from AF across the placenta into the mother's circulation. Alteration in the AF steroid hormone levels in cloned fetuses was associated with decreased expression of enzymes responsible for steroid hormone biosynthesis in the placenta. In conclusion, cloned pig fetuses undergo abnormal intrauterine development associated with alteration of bile acid and steroid hormone levels in AF, which may be due to the poor development of PFs and the erroneous expression of bile acid transporters and enzymes responsible for steroid hormone biosynthesis in the placentas.
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DOI:10.1002/mrd.22935URLPMID:29205617 [本文引用: 1]
The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow-derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear-transfer (BMSC-NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC-NT: 71.3 +/- 3.4%, 29.1 +/- 2.3%; IVF: 69.2 +/- 2.2%, 30.2 +/- 3.3%; respectively) than FF-NT embryos (58.1 +/- 3.4%, 15.1 +/- 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC-NT embryos (30.0%, 9.8%, respectively) than those in FF-NT embryos (34.2%, 28.0%, respectively). We also found that BMSC-NT embryos had more H3K9Ac and less H3K9me3 and 5-methylcytosine than FF-NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.
DOI:10.1016/s0093-691x(02)01294-3URLPMID:12600732 [本文引用: 1]
This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.
DOI:10.1002/mrd.20786URLPMID:17595009 [本文引用: 1]
Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos. The relative methylation in the gamete and blastocyst samples showed that 18.5% (921/4,992) of the DNA clones were found to be significantly different (P < 0.01) in at least one of the samples. Furthermore, for the different blastocyst groups, the methylation profile of the in vitro-produced blastocysts was less similar to the in vivo-produced blastocysts as compared to the parthenogenetic- and somatic cell nuclear transfer (SCNT)-produced blastocysts. The microarray results were validated by using bisulfite sequencing for 12 of the genomic regions in liver, sperm, and in vivo-produced blastocysts. These results suggest that a generalized change in global methylation is not responsible for the low developmental potential of blastocysts produced by using in vitro techniques. Instead, the appropriate methylation of a relatively small number of genomic regions in the early embryo may enable early development to occur.
DOI:10.1038/ng1973URLPMID:17325680 [本文引用: 2]
Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.
DOI:10.4161/epi.6.2.13519URL [本文引用: 1]
DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50 and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production.
DOI:10.1016/j.stem.2018.07.017URLPMID:30146410 [本文引用: 2]
Somatic cell nuclear transfer (SCNT) enables cloning of differentiated cells by reprogramming their nuclei to a totipotent state. However, successful full-term development of SCNT embryos is a low-efficiency process and arrested embryos frequently exhibit epigenetic abnormalities. Here, we generated genome-wide DNA methylation maps from mouse pre-implantation SCNT embryos. We identified widespread regions that were aberrantly re-methylated, leading to mis-expression of genes and retrotransposons important for zygotic genome activation. Inhibition of DNA methyltransferases (Dnmts) specifically rescued these re-methylation defects and improved the developmental capacity of cloned embryos. Moreover, combining inhibition of Dnmts with overexpression of histone demethylases led to stronger reductions in inappropriate DNA methylation and synergistic enhancement of full-term SCNT embryo development. These findings show that excessive DNA re-methylation is a potent barrier that limits full-term development of SCNT embryos and that removing multiple epigenetic barriers is a promising approach to achieve higher cloning efficiency.
DOI:10.1159/000494598URLPMID:30355946 [本文引用: 1]
BACKGROUND/AIMS: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. METHODS: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells' affects SCNT embryos development and the crosstalk between epigenetic signals. RESULTS: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3+/-1.4%, and 24.72+/-2.3%, respectively) than FF-SCNT embryos (60.1+/-2.4% and 18.38+/-1.9%, respectively). CONCLUSION: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.
DOI:10.1007/s10815-014-0320-2URL [本文引用: 2]
DOI:10.1111/age.12436URLPMID:27028246 [本文引用: 1]
Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer.
DOI:10.1186/1471-2164-15-811URL [本文引用: 2]
DOI:10.1371/journal.pone.0025901URLPMID:22022462 [本文引用: 1]
Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.
DOI:10.1016/j.bbagen.2008.12.006URLPMID:19168116 [本文引用: 1]
The eukaryotic DNA is wrapped around histone octamers, which consist of four different histones, H2A, H2B, H3 and H4. The N-terminal tail of each histone is post-transcriptionally modified. The modification patterns constitute codes that regulate chromatin organisation and DNA utilization processes, including transcription. Recent progress in technology development has made it possible to perform systematic genome-wide studies of histone modifications. This helps immensely in deciphering the histone codes and their biological influence. In this review, we discuss the histone modification patterns found in genome-wide studies in different biological models and how they influence cell differentiation and carcinogenesis.
DOI:10.1007/s00441-010-1000-xURL [本文引用: 1]
Swine play important roles as models of human disease. A combination of genetic modification with somatic cell nuclear transfer (SCNT) holds the promise of uncovering the pathogenesis of human diseases and then of developing therapeutic protocols. Unfortunately, the mechanism(s) of nuclear remodeling (a change in the structure of the nucleus) and reprogramming (a change in the transcriptional profile) during SCNT remains unclear. Incomplete remodeling is thought to cause lower cloning efficiency and abnormalities in cloned embryos and offspring. Here, we review the epigenetic regulatory and remodeling events that occur during preimplantation development of embryos derived from fertilization or SCNT, with a focus on DNA methylation and histone modifications. The discussion ends with a description of attempts at assisted remodeling of the donor somatic cell nucleus and the SCNT embryo.
DOI:10.1016/j.anireprosci.2014.06.002URL [本文引用: 1]
Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development. (C) 2014 Elsevier B.V.
DOI:10.1002/mrd.21180URLPMID:20422712 [本文引用: 1]
The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co-factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre-implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1- to the 4-cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1-cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4-cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4-cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst.
DOI:10.1262/jrd.20245URLPMID:19700928 [本文引用: 1]
Successful cloning by somatic cell nuclear transfer (SCNT) requires a reprogramming process in which the epigenetic state of a differentiated donor nucleus must be converted into an embryonic totipotent state. However, this epigenetic reprogramming is incomplete in SCNT embryos, causing low production efficiency. Recently, it has been reported that trichostatin A (TSA), an inhibitor of histone deacetylase, potentially enhances cloning efficiency. The aim of the present study was to optimize the TSA treatment for miniature pig SCNT embryos and investigate the effect of the acetylation level of histone on developmental competence of SCNT embryos. In order to optimize the TSA treatment, we examined the developmental competence of SCNT embryos under various exposure times (0-50 h) and concentrations (0-500 nM). Treatment with 5 nM TSA for 15 and 20 h beginning at the start of activation significantly increased the blastocyst formation rate (34.6 and 32.4 vs. 18.2%, respectively) and mean cell number (57.0 +/- 2.7 and 56.6 +/- 2.7 vs. 43.5 +/- 2.1, respectively) as compared with the non-treated group (0 h). We then investigated the acetylation levels of histone H3 in SCNT embryos treated with or without TSA (TSA (+) or TSA (-)) as compared with in vitro- fertilized (IVF) embryos. The acetylation levels of the TSA (-) SCNT embryos at the pseudo-pronuclear and 2-cell stages were significantly lower than those of the IVF embryos at the same developmental stages. In contrast, the acetylation levels of the TSA (+) SCNT embryos were similar to those of the IVF embryos. There was no difference in the acetylation levels of all groups at the blastocyst stage. Our data therefore suggests that the acetylation level of histone H3 at the pseudo-pronuclear and 2-cell stages is positively correlated with subsequent development of SCNT embryos, which may be an important event for the vital development of SCNT embryos in miniature pigs.
DOI:10.1371/journal.pone.0144897URLPMID:26683029 [本文引用: 1]
The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos.
DOI:10.1530/REP-15-0460URLPMID:26604326 [本文引用: 1]
Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.
DOI:10.1530/REP-15-0338URLPMID:26515777 [本文引用: 1]
Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 muM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 muM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.
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DOI:10.1126/science.1194174URLPMID:20847234 [本文引用: 1]
Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.
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DOI:10.1186/1756-0500-7-861URLPMID:25433481 [本文引用: 1]
BACKGROUND: Somatic cell cloning by nuclear transfer (SCNT) in pig is clearly of great benefit for basic research and biomedical applications. Even though cloned offspring have been successfully produced in pig, SCNT is struggling with the low efficiency. RESULTS: In the present study, we investigated differentially expressed proteins of the extraembryonic tissue from pig SCNT fetus compared to control (normal) fetus. We obtained the extraembryonic tissue from embryos at day 35 of pregnancy and examined the protein expression profiles using two-dimensional electrophoresis (2-D) and Western blotting. The extraembryonic tissue of fetus in control pregnancy was compared to the extraembryonic tissue of SCNT fetus, which showed an abnormally small size and shape as well as exhibited abnormal placental morphology compared to control fetus. A proteomic analysis showed that the expression of 33 proteins was significantly increased or decreased in the extraembryonic tissue of SCNT fetus compared to control fetus. The differentially expressed proteins in the extraembryonic tissue of SCNT fetus included ATP or lipid binding proteins, antioxidant proteins, translation elongation factors, and transcription factors. Western blotting analysis indicated that antioxidant enzymes and anti-apoptotic proteins were down-regulated; however, the expression levels of apoptotic proteins, Bax and Hsp27, were increased in the extraembryonic tissue of SCNT fetus. Moreover, immunohistochemical analysis also showed that the expression of the catalase or GPX genes was decreased in the extraembryonic tissue with SCNT fetus compared to those with control fetus. In addition, we observed a significant decrease in DNA methytransferase1 (Dnmt1) expression in SCNT extraembryonic tissue, and the expression levels of Dnmt3a and Dnmt3b were abnormally higher in SCNT fetus compared to control fetus. Moreover, a marked increase in the frequency of TUNEL-positive cells was observed in the extraembryonic tissue in SCNT fetus. CONCLUSION: These results demonstrated that pig SCNT fetus showed abnormal protein expression in the extraembryonic tissue, and extensive apoptosis occurred in the extraembryonic tissue of the SCNT fetus due to an increase in apoptotic protein expression or a decrease in antioxidant protein expression.
DOI:10.1002/mrd.23242URLPMID:31347235 [本文引用: 2]
Cloned pig fetuses produced by somatic cell nuclear transfer show a high incidence of erroneous development in the uteri of surrogate mothers. The mechanisms underlying the abnormal intrauterine development of cloned pig fetuses are poorly understood. This study aimed to explore the potential causes of the aberrant development of cloned pig fetuses. The levels of numerous fatty acids in allantoic fl uid and muscle tissue were lower in cloned pig fetuses than in artificial insemination-generated pig fetuses, thereby suggesting that cloned pig fetuses underwent fatty acid deficiency. Cloned pig fetuses also displayed trophoblast hypoplasia and a reduced expression of placental fatty acid transport protein 4 (FATP4), which is the predominant FATP family member expressed in porcine placentas. This result suggested that the placental fatty acid transport functions were impaired in cloned pig fetuses, possibly causing fatty acid deficiency in cloned pig fetuses. The present study provides useful information in elucidating the mechanisms underlying the abnormal development of cloned pig fetuses.
DOI:10.1016/j.theriogenology.2008.07.025URLPMID:18771798 [本文引用: 1]
The birth rate of cloned animals following somatic cell nuclear transfer (SCNT) is very low and the surviving animals have various developmental defects. We compared the morphology and transcriptional profile of extraembryonic tissue from three 26-d old SCNT pig fetuses with that from control fetuses. Transcriptional profiling using long-oligonucleotide microarray technology revealed 34 genes that were differentially expressed between the three groups. The differential expression of several genes involved in translational regulation was confirmed by real-time quantitative PCR and Western blot analysis. Interestingly, the expression of a translational inhibition-related gene encoding a eukaryotic translation initiation factor 4E-binding protein was significantly elevated in the SCNT samples. We concluded that the low birth rate of cloned animals could be related to abnormal expression of translational regulators in extraembryonic tissue during early pregnancy.
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DOI:10.1038/celldisc.2016.10URL [本文引用: 1]
DOI:10.1016/j.cell.2014.09.055URL [本文引用: 1]
Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.
DOI:10.15252/embr.201846240URLPMID:30389724 [本文引用: 1]
Despite the success of animal cloning by somatic cell nuclear transfer (SCNT) in many species, the method is limited by its low efficiency. After zygotic genome activation (ZGA) during mouse development, a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus-L (MuERVL/MERVL). In this study, we generate a series of MERVL reporter mouse strains to detect the ZGA event in embryos. We show that the majority of SCNT embryos do not undergo ZGA, and H3K27me3 prevents SCNT reprogramming. Overexpression of the H3K27me3-specific demethylase KDM6A, but not of KDM6B, improves the efficiency of SCNT Conversely, knockdown of KDM6B not only facilitates ZGA, but also impedes ectopic Xist expression in SCNT reprogramming. Furthermore, knockdown of KDM6B increases the rate of SCNT-derived embryonic stem cells from Duchenne muscular dystrophy embryos. These results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.
DOI:10.1016/j.stem.2015.10.001URLPMID:26526725 [本文引用: 1]
The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.
DOI:10.1242/dev.160382URLPMID:29361573 [本文引用: 1]
Stem cells (SCs) drive mammary development, giving rise postnatally to an epithelial bilayer composed of luminal and basal myoepithelial cells. Dysregulation of SCs is thought to be at the origin of certain breast cancers; however, the molecular identity of SCs and the factors regulating their function remain poorly defined. We identified the transmembrane protein podoplanin (Pdpn) as a specific marker of the basal compartment, including multipotent SCs, and found Pdpn localized at the basal-luminal interface. Embryonic deletion of Pdpn targeted to basal cells diminished basal and luminal SC activity and affected the expression of several Wnt/beta-catenin signaling components in basal cells. Moreover, Pdpn loss attenuated mammary tumor formation in a mouse model of beta-catenin-induced breast cancer, limiting tumor-initiating cell expansion and promoting molecular features associated with mesenchymal-to-epithelial cell transition. In line with the loss-of-function data, we demonstrated that mechanistically Pdpn enhances Wnt/beta-catenin signaling in mammary basal cells. Overall, this study uncovers a role for Pdpn in mammary SC function and, importantly, identifies Pdpn as a new regulator of Wnt/beta-catenin signaling, a key pathway in mammary development and tumorigenesis.
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DOI:10.1016/s0093-691x(02)01211-6URLPMID:12559468 [本文引用: 1]
In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos. In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS). Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%). Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05). In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts. The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope. The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted. In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored. No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups. A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%). The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%). When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media. Total cell number of blastocysts were not significantly different among experimental groups. In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.
DOI:10.1007/s12522-011-0079-4URL [本文引用: 1]
In most mammals, oocyte maturation is the final process of oogenesis, from the prophase of the first meiosis (germinal vesicle stage) to the metaphase of the second meiosis (MII), during which the oocyte acquires fertilizable competence as well as post-fertilization development competence. The nuclear and cytoplasmic maturation processes occur in synchrony but independently. Cytoplasmic maturation entails biochemical and structural changes in the cytoplasm, which give rise to oocytes capable of being fertilized and developing into embryos. Herein we review the literature and results from our own experiments on the structural and molecular events regulating cytoplasmic maturation in oocytes, concentrating on (1) the appropriate reorganization of active mitochondria and the endoplasmic reticulum, a structural and functional feature of cytoplasmic maturation, and (2) factors involved in regulatory mechanisms such as cumulus cell–oocyte gap junctional signaling, cumulus cell–oocyte bidirectional paracrine signaling, and the complex interactions of these signaling processes and follicular fluid constituents in the follicle environment.
DOI:10.1093/molehr/gax032URLPMID:28586460 [本文引用: 1]
STUDY QUESTION: Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? SUMMARY ANSWER: A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. WHAT IS KNOWN ALREADY: Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 x 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 +/- 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 +/- 7.4% versus 53.3 +/- 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 +/- 9%, 37.2 +/- 9.5% and 17.2 +/- 8.3%, respectively), although fetal development did not differ. LIMITATIONS, REASONS FOR CAUTION: Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. WIDER IMPLICATIONS OF THE FINDINGS: IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. LARGE SCALE DATA: N/A. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.
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DOI:10.1073/pnas.1703998114URL [本文引用: 1]
DOI:10.1089/cell.2019.0091URLPMID:32125895 [本文引用: 1]
Pig cloning technique is valuable in agriculture, biomedicine, and life sciences. However, the full-term developmental efficiency of cloned pig embryos is only about 1%, which limits pig cloning application. The quality of recipient oocytes greatly affects the developmental competence of cloned pig embryos. Thus, this study investigated the effects of a recipient oocyte source (in vivo matured [IVVM] oocytes vs. slaughter house-derived in vitro matured [IVTM] oocytes), and follicular liquid treatment (slaughter house-derived immature follicle-derived fluid [IFF] vs. in vivo-matured follicle-derived fluid [MFF]) during the in vitro maturation (IVM) of oocytes on the development of the cloned pig embryos. Our results showed that using IVVM oocytes to replace IVTM oocytes as recipient oocytes, and using 10% MFF IVM medium to replace 10% IFF IVM medium could enhance the development of the cloned pig embryos. IFF and MFF contained different levels of oocyte quality-related proteins, resulting in different oocyte quality-related gene expression levels and reactive oxygen species levels between the 10% MFF medium-cultured oocytes and 10% IFF medium-cultured oocytes. This study provided useful information for enhancing the pig cloning efficiency by improving the quality of recipient oocytes.
URLPMID:30786018 [本文引用: 1]
DOI:10.3390/molecules22122059URL [本文引用: 1]
DOI:10.1016/j.theriogenology.2010.05.026URLPMID:20688371 [本文引用: 1]
The aim of this study was to report from a larger study with pregnancy and delivery results after transfer of cloned transgenic/non-transgenic Large White or minipig embryos to Large White sow recipients. The effect of both total numbers of transferred embryos as well as site of their deposition (uni- vs. bi-lateral) was studied. Four to five days after natural heat, 85 Large White (LW) sows received Day 5 or 6 handmade cloned embryos. Large White embryos were non-transgenic and were transferred to 36 recipients, while 49 recipients each received Minipig embryos, either non-transgenic or with 1 of 4 types of transgenes. Furthermore, the number of embryos transferred was in two categories, as 46 recipients received 40-60 embryos while 39 received 60-120 embryos. Finally, in 59 of the recipients embryos were transferred to one of the uterine horns (unicornual) while 26 other recipients had embryos transferred to both uterine horns (bicornual). The overall pregnancy rate was 55% with an abortion rate of 26% resulting in 41% deliveries with no difference between LW and Minipig embryos and no difference between transgenic and non-transgenic Minipig embryos. Transfer of 60-120 embryos resulted in more pregnancies and deliveries (62%) than <60 embryos (24%). The mean litter size was 5.1 +/- 0.5 and after transfer of 60-120 embryos significantly higher (6.0 +/- 0.5) than after transfer of <60 embryos (3.5 +/- 0.8). Also, the bicornual transfer resulted in significantly higher delivery rate (74% vs. 44%) and mean litter size (6.1 +/- 0.7 vs. 4.2 +/- 0.6) than the unicornual. The mean rate of piglets/transferred embryos was 7.3 +/- 0.6% while the mean rate of piglets/reconstructed embryos was 179/18,000 = 1% with no difference between breeds or number of embryos transferred. The overall perinatal mortality rate was 49%, and it was significantly lower in LW piglets (20/59 = 34%) than in Minipiglets (67/120 = 56%) (vs. 10-15% in normal piglets at the farm) and the total rate of piglets with one or more malformation was 22%, and lower in LW (12%) than in Minipiglets (28%). This study demonstrate that although the perinatal mortality was rather high, an acceptable birth rate can be achieved after transfer to LW recipients of cloned LW embryos as well as cloned, transgenic/non-transgenic Minipig embryos. Furthermore, the pregnancy rate and litter size were correlated to the number of embryos transferred and to bicornual transfer.
DOI:10.1016/j.anireprosci.2015.01.006URLPMID:25640459 [本文引用: 1]
The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs.
DOI:10.1089/clo.2008.0026URLPMID:18729768 [本文引用: 1]
The efficiency of porcine somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.
DOI:10.1073/pnas.1814514115URLPMID:30381455 [本文引用: 1]
Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.
DOI:10.1016/j.anireprosci.2018.08.013URLPMID:30115522 [本文引用: 1]
The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14-75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.
DOI:10.1016/j.cell.2018.02.028URL [本文引用: 1]
DOI:10.1016/j.cell.2016.05.050URLPMID:27259149 [本文引用: 1]
How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos.
URLPMID:29915360 [本文引用: 1]
DOI:10.1038/nature18606URLPMID:27309802 [本文引用: 1]
In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development.
DOI:10.1038/nature12593URL [本文引用: 1]
Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.
DOI:10.1016/j.stem.2018.06.018URLPMID:30033121 [本文引用: 1]
Successful cloning of monkeys, the first non-human primate species, by somatic cell nuclear transfer (SCNT) attracted worldwide attention earlier this year. Remarkably, it has taken more than 20 years since the cloning of Dolly the sheep in 1997 to achieve this feat. This success was largely due to recent understanding of epigenetic barriers that impede SCNT-mediated reprogramming and the establishment of key methods to overcome these barriers, which also allowed efficient derivation of human pluripotent stem cells for cell therapy. Here, we summarize recent advances in SCNT technology and its potential applications for both reproductive and therapeutic cloning.
DOI:10.1016/j.stem.2017.12.001URLPMID:29358044 [本文引用: 1]
Generation of induced pluripotent stem cells typically requires the ectopic expression of transcription factors to reactivate the pluripotency network. However, it remains largely unclear what remodeling events on endogenous chromatin trigger reprogramming toward induced pluripotent stem cells (iPSCs). Toward this end, we employed CRISPR activation to precisely target and remodel endogenous gene loci of Oct4 and Sox2. Interestingly, we found that single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network. Simultaneous remodeling of the Oct4 promoter and enhancer also triggered reprogramming. Authentic pluripotent cell lines were established in both cases. Finally, we showed that targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming. Our study generated authentic iPSCs with CRISPR activation through precise epigenetic remodeling of endogenous loci and shed light on how targeted chromatin remodeling triggers pluripotency induction.
DOI:10.1016/j.cell.2016.08.056URLPMID:27662091 [本文引用: 1]
Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.
