,华南农业大学动物科学学院,国家生猪种业工程技术研究中心,广州 510642Advances in epigenetic reprogramming of somatic cells nuclear transfer in mammals
Xuqiong Yang, Zhenfang Wu, Zicong Li,National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China通讯作者: 李紫聪，教授，博士生导师，研究方向：动物遗传育种与繁殖。E-mail:lizicongcong@163.com
编委: 高绍荣
收稿日期:2019-07-3修回日期:2019-10-7网络出版日期:2019-12-20
| 基金资助: |
Editorial board:
Received:2019-07-3Revised:2019-10-7Online:2019-12-20
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作者简介 About authors
杨旭琼,硕士研究生,专业方向：动物遗传育种与繁殖。E-mail:1814639793@qq.com。
摘要
体细胞核移植(somatic cell nuclear transfer, SCNT)是唯一能赋予体细胞基因组全能性的生殖工程技术,对动物种质资源保存、畜牧业发展和生物医学研究等具有重大意义。尽管该技术已经取得了许多研究进展,但哺乳动物克隆胚胎的发育效率依然很低,严重限制其在畜牧业和生物医学上的应用。导致克隆胚胎发育效率低的主要原因是体细胞重编程错误或重编程不完全,主要表现为：印记基因Xist表达异常、DNA甲基化异常,组蛋白修饰异常等。本文简要介绍了体细胞核移植技术,系统总结了哺乳动物克隆胚胎发育效率低的主要影响因素,以期为提升体细胞克隆效率相关研究与实践提供理论参考。
关键词：
Abstract
Somatic cell nuclear transfer (SCNT) is the only reproductive engineering technique that can confer genomic totipotency on somatic cell. SCNT is of great significance for animal germplasm conservation, animal husbandry development, and biomedical research. Although many research advances have been made in this technology, the developmental rate of SCNT mammalian embryos is very low, which seriously limits the application of SCNT in animal husbandry and biomedicine. The primary reason for the low efficiency of cloned embryos is somatic cell reprogramming errors or incomplete reprogramming. These errors or incompleteness present as the abnormal expression of imprinted gene Xist, abnormal DNA methylation, and abnormal histone modification. In this review, we summarize the main factors that influence the low development efficiency of mammalian cloned embryos to provide theoretical reference for the research and practice of improving somatic cell cloning efficiency.
Keywords：
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本文引用格式
杨旭琼, 吴珍芳, 李紫聪. 哺乳动物体细胞核移植表观遗传重编程研究进展. 遗传[J], 2019, 41(12): 1099-1109 doi:10.16288/j.yczz.19-193
Xuqiong Yang.
体细胞核移植(somatic cell nuclear transfer, SCNT)又称体细胞克隆,指利用显微操作技术,以去核卵母细胞为受体,单细胞核为供体,将体细胞核移入成熟的去核卵母细胞中,激活形成克隆胚胎,进而培育出基因型与供体体细胞相同的克隆动物(图1)。体细胞核移植技术是当代生命科学研究和应用的关键技术之一,是生命科学研究高水平发展的体现,在农业动物生产、药物生产、再生医学和保护宝贵遗传资源等方面具有广泛的应用价值。然而,克隆胚胎的发育效率远远低于体外受精胚的发育效率,哺乳动物的克隆胚胎发育效率只有1%~5%[1],严重限制了克隆技术的发展。导致体细胞克隆效率低下的主要原因是体细胞重编程错误或重编程不完全[2,3]。鉴于此,研究人员希望通过一些有效的技术手段来提高克隆胚胎的发育能力,如采用不同的细胞系作为供体细胞,同时优化克隆操作的融合参数[4,5,6,7]、敲除印记基因Xist (X-inactive specific transcript)[8]、RNA干扰技术(RNA interference, RNAi)抑制Xist基因的异常表达[9]、注射Kdm4b或Kdm5b去甲基化酶[10,11]等。这些研究虽然取得了一定的效果,但大多数研究结果表明克隆效率并没有大幅度的提高,且克隆动物后代存在诸多异常。因此,如何克服克隆效率低下和解决克隆动物异常成为体细胞克隆研究的热点。
体细胞克隆的供体细胞是高度分化的体细胞,具有高度特异的DNA修饰和组蛋白修饰,以此来维持体细胞的细胞特性。由于克隆胚胎的发育依赖于体细胞的细胞核,所以当供体细胞核被放置到成熟的去核卵母细胞中时,供体细胞核必须经过重编程,抹去分化状态的细胞记忆,激活对早期胚胎发育具有重要作用的基因,如多能性基因、抑制与分化相关的基因,从而使得体细胞获得发育的全能性[12]。由于重编程发生在一个相对较短的时间框架内,若克隆胚胎的发育与正常的胚胎发育不一致,克隆胚胎的发育状态会出现一系列的错误。越来越多的数据表明,错误的重编程模式会使克隆动物出现表观遗传修饰的偏差。如X染色体失活[13]、印记基因与非印记基因的表达[3,12~14]、DNA甲基化[15,16,17]和染色体修饰[18]等。本文对体细胞核移植技术的发展以及影响克隆胚胎发育效率低的主要原因进行了总结,以期为未来提高哺乳动物克隆发育效率的研究提供参考。
1 体细胞核移植技术
1.1 体细胞核移植技术的发展历程
SCNT技术实现了体细胞的全能性。早在1952年,英国遗传学家Briggs和King将青蛙(Xenopus laevis)胚胎卵裂球细胞的细胞核移植到去核的卵母细胞中,以此来研究胚胎干细胞的细胞核是否发生分化。这是首次利用两栖动物的胚胎干细胞实现胚胎细胞核移植技术,但是当时并未成功克隆出青蛙[19]。1962年,英国生物学家Gurdon首次在两栖动物上利用SCNT技术成功地将分化的青蛙体细胞克隆出小蝌蚪[20]。直到1997年第一头克隆羊“多莉”(Dolly)诞生[21],这是世界上第一个克隆成功的哺乳动物。随后,奶牛(Bos taurus)[22]、小鼠(Mus musculus)[23]、山羊(Capra hircus)[24]、猪(Sus scrofa)[25,26]、兔子(Oryctolagus cuniculus)[27]、猫(Felis catus)[28]、骡子(Equus ferus x asinus)[29]、马(Equus caballus)[30]、大鼠(Rattus norvegicus)[31]、猎犬(Canislupus familiaris)[32]和骆驼(Camelus bactrianus)[33]等成功克隆的20多种哺乳动物相继问世。2017年,我国成功克隆出世界上第一批体细胞克隆猴(Macaca mulatta)[34]。图1
新窗口打开|下载原图ZIP|生成PPT图1体细胞核移植的流程
Fig. 1Process of somatic cell nuclear transfer
1.2 体细胞核移植技术的应用和存在问题
SCNT技术能够培育优良畜种,如选育高品质家畜和扩大繁殖高性能产量个体;此外还可以培育抗病物种、制备异种器官移植供体动物、制备人类疾病动物模型和转基因动物生物反应器等。除了克隆动物外,SCNT在干细胞生物学和人类疾病治疗等方面具有巨大的潜力。受精卵发育到囊胚期分化形成内细胞团(inner cell mass, ICM)和滋养层细胞(trophoblast, TE),其中ICM可分离培养出胚胎干细胞(embryonic stem cells, ESCs)。克隆胚胎发育到囊胚期,ICM也可分离培养出多能干细胞(pluripotent stem cells, PSCs)或者称核移植胚胎干细胞(nuclear transfer embryonic stem cells, ntESCs)[35]。在生物医学疾病治疗方面,治疗性克隆能够通过培养人的ntESCs,建立并保存每个个体自身的ntESCs,用于组织和器官替代疗法。患者的ntESCs和患者本身具有相同的基因组,可避免排斥反应等不适问题[36,37]。2001年,Wakayama等[38]在成年小鼠体细胞克隆胚胎中分离出了具备多能性特征的核移植ESCs,这也是第一例ntESCs,为人ntESCs的研究提供了实验基础。Rideout等[39]通过同源重组的方法实现了突变等位基因的遗传固定,并获得ntESCs细胞系用作治疗免疫缺陷小鼠。2007年,Byrne等[40]成功获得猴子的ntESCs。2013年,Tachibana等[35]获得了第一例人的ntESCs。随后,更多的实验室相继报道获得了健康成年人[41]、糖尿病[37]及老年性黄斑变性病[36]人体细胞来源的ntESCs。众所周知,ntESCs在人类中的研究能为组织和器官功能失调或受损的患者提供干细胞新来源。这种干细胞可以更新和替换损坏了的细胞或组织,可为上百万的患者减缓病情。在临床上用患有线粒体疾病患者的卵细胞核移植到另一个健康去核卵细胞中,从而阻断线粒体疾病的下一代遗传。2017年张进等[42]利用Leigh氏综合征携带者卵细胞纺锤体移植获得了健康婴儿,这是第一例线粒体替代婴儿。当然,线粒体疾病的替代治疗存在人们关心的伦理问题,这也是阻碍其临床广泛推广的主要原因。
目前,SCNT技术已经成熟,但是依然存在一些问题,严重限制其在生产实际中的应用和发展。总的来说,哺乳动物的克隆效率都比较低下,主要表现在：克隆胚胎体外发育效率低,如猴SCNT单个卵母细胞的孵化效率仅为0.7%[40];体内发育至出生效率低,例如猪的出生率大约在0.5%~1%左右[43,44]。在克隆胚胎中,由TE分化形成的胎盘经常存在异常状态[45],胎盘异常几乎是所有克隆哺乳动物的一个共有特征,例如胎盘增生、胎盘血管缺陷、脐带畸形[46]等。此外,克隆动物的健康状况也存在一定的异常,包括肥胖、免疫及呼吸缺陷和早期死亡等[45,47,48]。由于SCNT技术受到卵母细胞、供体细胞的质量以及代孕母体等个体差异的影响,因此很难从统计学的数据分析上确定影响因素[49]。
2 表观遗传重编程对体细胞核移植胚胎发育效率的影响
生物体的大多数细胞具有相同的遗传物质,SCNT重编程主要通过表观遗传重编程来实现。在克隆胚胎发育早期,存在体细胞标记和细胞类型特异性分化记忆。无论是体细胞标记还是细胞特异性分化记忆都可能导致特定的重编程错误,致使在克隆胚胎发育过程中出现各种异常。若要使其正常发育,克隆胚胎应该以某种方式克服这两个表观遗传障碍。因此,当供体细胞核与去核的卵母细胞融合后,供体细胞核需要对核内已有的表观遗传修饰进行重编程,即擦除供体细胞表观遗传模式,激活与胚胎发育相关的基因,抑制与细胞分化相关的基因,重新获得发育的全能性。当胚胎附植于子宫后,胚胎从全能状态再分化,用于组织生成及器官发生[50]。而在克隆胚胎重编程的过程中,由于体细胞的表观遗传修饰去除不完全,未能建立起正确的表观遗传修饰来调控胚胎的正常发育,致使其出现各种异常。表观遗传重编程主要包括基因组印记、X染色体失活、DNA甲基化和组蛋白修饰等(表1)。Table 1
表1
表1小鼠和猪SCNT胚胎发育效率的表观遗传重编程影响因素及对策
Table 1
| SCNT胚胎发育效率[44,45,47,50,94] | 影响SCNT胚胎发育效率 的表观遗传重编程因素 | 对策 | 结果 | |
|---|---|---|---|---|
| 体外(囊胚率) | 体内(出生率) | |||
| 15% ~ | 1%~5% | Xist | KO-Xist;注射siRNA | 克隆小鼠出生率提高8~9倍[8]; 克隆小鼠囊胚率以及出生率提高10倍[9];克隆猪出生率提高6.9倍[64] |
| DNA甲基化 | 抑制DNMT | 克隆小鼠出生率提高5倍[75] | ||
| 组蛋白乙酰化水平 | 添加HDACi药物 | 克隆猪囊胚率提高2倍[81]; 克隆小鼠囊胚率提高5~10倍[80] | ||
| H3K9me3 | 注射Kdm4d或联合KO-Xist; 联合注射Kdm4b和Kdm5b | 克隆小鼠出生率提高13~16倍[74]; 克隆小鼠囊胚率达95%[10] | ||
| H3K27me3 | 注射Kdm6a | 克隆小鼠囊胚率提高2倍[95] | ||
新窗口打开|下载CSV
2.1 抑制印记基因Xist表达可显著提高体细胞核移植胚胎发育效率
XY型哺乳动物,其X和Y染色体是由同源常染色体进化而来。由于雄性和雌性X染色体上的基因数目不同,两者之间存在大规模的遗传失衡,为平衡这种剂量差异,在雌性胚胎发育过程中会选择失活其中一条X染色体[51]。Xist基因是X染色体上顺式调控X染色体失活的印记基因,其转录产物是在X染色体失活中心(X-chromosome inactivation, XCI)开始转录的长链非编码RNA(long non-coding RNA, lncRNA),转录产物lncRNA通过包围整条X染色体使得X染色体失活[52]。哺乳动物X染色体存在两种形式的失活方式：印记失活和随机失活。XCI在胚胎发育早期建立,受胚胎发育调控,且调控方式与物种种类密切相关[53]。雌性小鼠胚胎发育过程中,在胚胎2~4细胞期Xist基因从父源X染色体上开始转录启动表达,致使父方来源X染色体失活,此印记表达模式,在胚胎外组织中始终维持[54]。当胚胎发育到囊胚期时,这种印记形式的X染色体失活会在ICM中经历再活化,直至胚胎着床期,ICM中会随机失活一条X染色体[55]。但是,人类胚胎发育中Xist表达模式并没有像小鼠一样,而是在胚胎发育后期随机失活[56]。
动物克隆胚胎发育过程中存在许多表达异常的基因,Xist是其中之一[57]。印记基因Xist的表达与表观遗传修饰乙酰化、甲基化密切相关,包括组蛋白H3和H4低乙酰化、H3-lysine 4(H3K4)低甲基化、H3-lysine 9 (H3K9)甲基化和多梳沉默复合体(polycomb repressive complex 2, PRC2)依赖的H3-lysine 27 (H3K27)甲基化等[58,59]。比较克隆胚胎和受精胚胎的转录产物,发现无论雌性或雄性小鼠克隆胚胎中Xist基因都异常表达,其X染色体连锁基因都受到持续地特异性抑制,导致染色体水平的基因大面积下调[8]。同样,Fukuda等[60]发现小鼠克隆胚胎中Xist异常表达,X染色体异常失活。研究表明,Xist在小鼠克隆胚胎桑葚胚期开始异常高表达,结果导致了染色体水平的大面积基因下调,利用Xist基因缺陷型供体细胞用于克隆实验,可显著提高小鼠克隆效率,克隆效率提高到8~9倍[8]。在雄性小鼠克隆胚胎中注入抗Xist的小干扰RNA (siRNA),也观察到了类似的效果,同时也表明克隆胚胎植入前Xist的异常表达严重影响克隆胚胎的发育能力[9]。敲除供体细胞的Xist基因或干扰克隆胚胎中的Xist基因显著的提高了小鼠的克隆效率,这说明纠正Xist基因的错误表达对克隆胚胎的发育效率有显著作用。
对猪而言,发育异常的克隆胎儿同样存在Xist的异常表达,且这种异常始于桑椹胚期[61]。通过RNAi的方式,在克隆胚胎1-细胞期注射siRNA,结果表明猪克隆胚的Xist表达异常升高,小幅度提高了猪克隆胚胎发育效率[57]。一方面,由于克隆所用供体细胞是猪肾髓质部细胞,该细胞易老化,易发生癌变,致使Xist表达异常升高;另一方面,因为siRNA作用时间太短,当猪克隆胚胎发育到桑椹胚时,siRNA已经失去它的干扰作用,所以通过注射siRNA提高猪克隆胚胎的发育效率似乎并不可行。此外,通过RNAi的方式干扰Xist基因,对提高猪孤雌胚胎发育效率也有显著效果[62]。Yang等[63]利用TALEN技术突变猪供体细胞的Xist基因,结果表明X染色体部分再活化,并没有提高猪克隆效率。Ruan等[64]利用TALEN技术在猪供体细胞Xist基因第一外显子重复序列前以插入大片段的方式破坏重复序列,进而失活Xist基因,大幅度提高了猪的克隆发育效率。但是,出生猪只数较少,共移植530个猪克隆胚胎,获得健康克隆胎儿11只。
2.2 DNA去甲基化水平影响体细胞核移植胚胎发育效率
哺乳动物DNA甲基化是指在DNA甲基化转移酶(DNA methyltransferase, DNMT)的帮助下,将DNA分子中S-腺苷蛋氨酸的甲基转移至胞嘧啶残基的第5位碳原子上的过程[65,66,67]。DNA甲基化由DNMT建立和维持,相反地,TET蛋白酶(ten-eleven translocation, TET)可以催化5-mC (5-methylcytosine, 5-mC)转化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5-hmC),进而启动DNA去甲基化程序[68,69]。哺乳动物早期胚胎发育,基因组中的DNA甲基化修饰会发生重编程过程,广泛进行去甲基化,以此在囊胚期达到最低水平。牛、鼠、猪等动物受精后,父本和母本经历不同的去甲基化方式,前者主动去甲基化,后者被动去甲基化[16,70]。DNA去甲基化是细胞多能性建立和维持的关键步骤,是重编程的第一步,同时也是核移植后早期胚胎发育正常启动和维持的重要环节[71],对表观遗传修饰起到关键作用。体细胞基因组的CpG岛大多数处于高度甲基化状态,全面去甲基化则是SCNT重编程的必须步骤[72]。相比正常的体外受精胚胎,克隆胚胎基因组去甲基化也发生在卵裂时期,但是克隆胚胎去甲基化不完全,其基因组甲基化水平更接近体细胞的状态[50]。Inoue等[73]研究发现,在小鼠受精胚胎中,除印记基因外,新合成的DNA大多未被甲基化。然而,Matoba等[74]研究发现在小鼠克隆胚胎囊胚期,某些基因的启动子部位具有高水平的DNA甲基化。Gao等[75]研究表明异常的DNA再甲基化阻碍了合子基因组激活,是影响SCNT胚胎发育的重要表观遗传障碍。DNMT的抑制能够克服DNA再甲基化的缺陷,同时提高植入后SCNT胚胎发育效率及克隆效率,抑制DNMT和过表达组蛋白赖氨酸去甲基化酶(K-demethylases, Kdms)相结合的方法可以进一步提高克隆效率。以上研究表明,DNA甲基化程度是重要的表观遗传障碍之一。2.3 组蛋白修饰可改善体细胞核移植胚胎发育效率
组蛋白脱乙酰酶(histone deacetylase, HDAC)可调节组蛋白的乙酰化水平,从而实现对基因表达的表观遗传调控[76]。组蛋白脱乙酰酶抑制剂(histone deacetylase inhibitor, HDACi)通过其功能基团与HDAC的Zn2+形成螯合物,抑制HDAC的活性,增加细胞内组蛋白的乙酰化程度,从而提高靶基因的表达水平[76]。HDACi在动物克隆胚胎发育中被广泛用来改善不同物种胚胎发育的重编程[77]。早在2006年Kishigami等[78]和Rybouchkin等[79]发现HDACi能使小鼠克隆胚胎效率从1%提高到6%。曲古抑菌素A (trichostatin A, TSA)是一种有效的HDACi。Inoue等[80]通过添加TSA药物处理小鼠克隆胚胎,显著提高了克隆胚胎2-细胞期后的发育效率,从而将克隆效率提高了5~10倍,但是TSA对克隆胚胎中异常表达的基因数量以及表达模式没有影响。同样,HDACi药物治疗使得猪[31,81,82]、牛[83,84,85]的克隆胚胎发育效率均有所提高,但其对SCNT重编程的机制仍不清楚。此外,也有研究表明用HDACi处理猪克隆胚胎,H3K14、H4K5和H4K8等赖氨酸残基出现乙酰化现象[86,87]。哺乳动物的卵母细胞和精子本身处于转录沉默,受精后,受精卵则恢复转录,该过程称为合子基因组激活(zygotic genome activation, ZGA)。不同物种的ZGA时间不同,小鼠和人的ZGA时间分别在胚胎2-细胞期和胚胎8-细胞期。当ZGA启动时,受精胚胎中母系储存的RNA迅速降解,并被新合成的合子RNA取代。同样地,克隆胚胎的ZGA也存在类似机制,且克隆胚胎早期发育过程中出现发育停滞的时间与ZGA时间高度相关。有研究表明,在小鼠克隆胚胎中大约有1000个基因组区域或基因未能在ZGA时间内激活[11]。有趣的是,在重编程抵抗区(reprogramming-resistant regions, RRRs)富含转录抑制标记物H3K9me3,这说明了供体细胞中组蛋白H3K9me3可能是阻止克隆胚胎ZGA的屏障。Matoba等[11]通过注射H3K9me3特异性去甲基酶Kdm4d的mRNAs不仅克服了ZGA缺陷,而且解决了植入前胚胎发育停滞的问题,使得幼崽出生率提高了8%以上。
Wang等[88]揭示了异染色质组蛋白修饰H3K9me3在配子细胞以及受精后和早期胚胎发育过程中的重编程与其在逆转座子沉默中的作用及调控机制。相关研究表明,供体细胞和2-细胞期克隆胚胎中在某些区域都富含异染色质组蛋白H3K9me3标记[89],且在小鼠克隆胚胎2-细胞期,存在一些区域没有去甲基化[10]。这一观察结果也证实了供体细胞中H3K9me3是SCNT重编程的表观遗传屏障[11,36,44]。研究表明在小鼠克隆胚胎2-细胞期和4-细胞期注射Kdm4b和Kdm5b去甲基化酶,针对组蛋白H3K9me3和H3K4me3去甲基化,显著提高了囊胚发育率,且从克隆胚胎中成功分离培养出ntESCs[10]。Matoba等[74]采用敲除Xist (KO-Xist)供体细胞与Kdm4d- mRNA注射相结合的方法,以支持细胞作为供体细胞克隆小鼠,使得克隆效率显著提高到24%。尽管如此,小鼠克隆效率依然低于体外受精发育效率。最近研究表明,通过注射Kdm4b也可以提高猪[64]、牛[90]以及猴[34]的克隆效率。以上结果说明,H3K9me3去甲基化是克隆胚胎正常发育过程中重编程所必需的组蛋白修饰,同时也是克隆胚胎成功重编程的限制因素之一[91]。此外,母源印记H3K27me3组蛋白修饰同样是影响克隆胚胎重编程的重要因素。研究发现,受精胚胎调控印记基因的母源H3K27me3结构域并未在克隆胚胎中建立[92,93],致使H3K27me3依赖性印记基因大部分失去其印记状态,成为双等位基因表达[74]。Inoue等[93]表示印记基因Xist也受母源H3K27me3的调控,由于供体细胞的Xist位点缺少H3K27me3标记,克隆胚胎中H3K27me3的重编程不完全,进而导致克隆胚胎Xist异常激活。因此,为解决供体细胞中H3K27me3的缺失问题,在供体细胞母源等位基因中靶向沉积H3K27me3可能是一个必要的策略。
3 结语与展望
SCNT的成功是生命科学领域的一次重大突破,其在优良种畜扩繁、濒危物种保护、克隆性治疗等方面具有广阔的应用前景。然而,运用克隆技术成功克隆出青蛙距今已有50余年,克隆胚胎发育至成体的成功率仍保持在一个很低水平。尽管,自Dolly羊诞生20年来,科学家致力于SCNT操作过程中影响克隆胚胎发育效率的各种条件和参数的研究,但克隆效率并未得到显著提高。克隆效率低的根本原因是供体细胞核的表观重编程异常[50]。对此,人们需要对重编程过程中染色质和表观基因组的变化进行系统和详细的分析。随着测序技术的更新换代,转录组测序及相关的表观遗传学研究,使得对SCNT的重编程研究成为可能[96]。从技术上来说,获取足够的克隆样本用于此类分析仍然具有较高的难度,但近些年的相关研究证明,利用早期胚胎进行此类研究具有一定的可行性[57,97,98]。SCNT可将分化的体细胞重编程为全能性胚胎,但在克隆胚胎早期发育过程中,大多数克隆胚胎会出现停滞现象,其潜在的分子机制尚未明了。科研人员对提高克隆效率的研究,使得表观遗传障碍与其特定的重编程错误两者之间的关系变得更加清晰,从而更加准确地理解在细胞分化和克隆胚胎植入过程中,表观遗传调节机制的作用。此外,通过比较分析不同重编程系统之间的异同,来探究克隆胚胎的重编程机制也是一种可行的方案。例如,H3K9me3组蛋白修饰、染色质组装因子(CAF1)蛋白质复合物、异染色质蛋白1 (HP1)是诱导多能干细胞(induced pluripotent stem cells, iPSCs)重编程的障碍[99,100,101],而iPSCs重编程与SCNT的重编程机制类似。对此,在今后的研究中,进一步探究这些重编程障碍是否也在SCNT重编程中起作用,可以作为体细胞核移植潜在的研究方向。总之,供体核的表观重编程异常修复依然是体细胞核移植研究及发展的重点。利用新型技术,如高通量测序[102,103]、CRISPR/Cas9[104,105]等,将更加快速准确地解析体细胞表观重编程机制,从而大幅度提高克隆效率,降低克隆动物异常表型的发生率,最终将SCNT技术应用于更多领域。
参考文献 原文顺序
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被引期刊影响因子
DOI:10.1038/ng1895URLPMID:17013394 [本文引用: 1]
Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.
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:10.1126/science.1063206URLPMID:11498580 [本文引用: 2]
Cloning of mammals by nuclear transfer (NT) results in gestational or neonatal failure with at most a few percent of manipulated embryos resulting in live births. Many of those that survive to term succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Cloned embryos derived from donors, such as embryonic stem cells, that may require little or no reprogramming of early developmental genes develop substantially better beyond implantation than NT clones derived from somatic cells. Although recent experiments have demonstrated normal reprogramming of telomere length and X chromosome inactivation, epigenetic information established during gametogenesis, such as gametic imprints, cannot be restored after nuclear transfer. Survival of cloned animals to birth and beyond, despite substantial transcriptional dysregulation, is consistent with mammalian development being rather tolerant to epigenetic abnormalities, with lethality resulting only beyond a threshold of faulty gene reprogramming encompassing multiple loci.
DOI:10.1095/biolreprod63.2.513URLPMID:10906058 [本文引用: 1]
Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.
URLPMID:11058438 [本文引用: 1]
Twenty-four calves were cloned from six somatic cell types of female and male adult, newborn and fetal cows. The clones were derived from female cumulus (n = 3), oviduct (n = 2) and uterine (n = 2) cells, female and male skin cells (n = 10), and male ear (n = 5) and liver (n = 2) cells. On the basis of the number of cloned embryos transferred (n = 172) to surrogate cows, the overall rate of success was 14%, but based on the number of surrogate mothers that became pregnant (n = 50), the success rate was 48%. Cell nuclei from uterus, ear and liver cells, which have not been tested previously, developed into newborn calves after nuclear transfer into enucleated oocytes. To date, seven female and six male calves have survived: six of the females were from adult cells (cumulus (n = 3), oviduct (n = 2) and skin (n = 1) cells) and one was from newborn skin cells, whereas the male calves were derived from adult ear cells (n = 3), newborn liver and skin cells (n = 2), and fetal cells (n = 1). Clones derived from adult cells frequently aborted in the later stages of pregnancy and calves developing to term showed a higher number of abnormalities than did those derived from newborn or fetal cells. The telomeric DNA lengths in the ear cells of three male calves cloned from the ear cells of a bull aged 10 years were similar to those of the original bull. However, the telomeric DNA lengths from the white blood cells of the clones, although similar to those in an age-matched control, were shorter than those of the original bull, which indicates that telomeric shortening varies among tissues.
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:10.1089/clo.2006.0002URLPMID:17386005 [本文引用: 1]
DOI:10.1126/science.1194174URLPMID:20847234 [本文引用: 4]
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.
DOI:10.1073/pnas.1112664108URLPMID:22065773 [本文引用: 3]
Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.
DOI:10.1038/celldisc.2016.10URLPMID:27462457 [本文引用: 4]
Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos.
DOI:10.1016/j.cell.2014.09.055URL [本文引用: 4]
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:10.1073/pnas.192433399URLPMID:12235366 [本文引用: 2]
To assess the extent of abnormal gene expression in clones, we assessed global gene expression by microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived by nuclear transfer (NT) from both cultured embryonic stem cells and freshly isolated cumulus cells. Direct comparison of gene expression profiles of more than 10,000 genes showed that for both donor cell types approximately 4% of the expressed genes in the NT placentas differed dramatically in expression levels from those in controls and that the majority of abnormally expressed genes were common to both types of clones. Importantly, however, the expression of a smaller set of genes differed between the embryonic stem cell- and cumulus cell-derived clones. The livers of the cloned mice also showed abnormal gene expression, although to a lesser extent, and with a different set of affected genes, than seen in the placentas. Our results demonstrate frequent abnormal gene expression in clones, in which most expression abnormalities appear common to the NT procedure whereas others appear to reflect the particular donor nucleus.
DOI:10.1038/ng900URLPMID:12032569 [本文引用: 1]
In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.
DOI:10.1089/153623002753632020URLPMID:12006154 [本文引用: 1]
Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.
DOI:10.1016/s0960-9822(01)00480-8URLPMID:11591324 [本文引用: 1]
Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.
DOI:10.1073/pnas.241522698URLPMID:11717434 [本文引用: 2]
Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.
DOI:10.1038/88903URLPMID:11381267 [本文引用: 1]
Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.
DOI:10.1016/S0960-9822(03)00419-6URL [本文引用: 1]
Abstract
During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state [1]. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development [2], [3], [4], [5] and [6]. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos [7], [8], [9], [10], [11] and [12]. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer [7], [8], [9], [10], [11] and [12]. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.DOI:10.1073/pnas.38.5.455URLPMID:16589125 [本文引用: 1]
URLPMID:13951335 [本文引用: 1]
DOI:10.1038/385810a0URLPMID:9039911 [本文引用: 1]
Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
DOI:10.1126/science.282.5396.2095URLPMID:9851933 [本文引用: 1]
Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.
DOI:10.1038/28615URLPMID:9690471 [本文引用: 1]
Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.
DOI:10.1038/8632URLPMID:10331804 [本文引用: 1]
In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.
DOI:10.1038/35024082URLPMID:10993078 [本文引用: 1]
Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.
DOI:10.1126/science.289.5482.1188URLPMID:10947985 [本文引用: 1]
Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.
DOI:10.1038/nbt0402-366URLPMID:11923842 [本文引用: 1]
We have developed a method to produce live somatic clones in the rabbit, one of the mammalian species considered up to now as difficult to clone. To do so, we have modified current cloning protocols proven successful in other species by taking into account both the rapid kinetics of the cell cycle of rabbit embryos and the narrow window of time for their implantation after transfer into foster recipients. Although our method still has a low level of efficiency, it has produced several clones now proven to be fertile. Our work indicates that cloning can probably be carried out successfully in any mammalian species by taking into account physiological features of their oocytes and embryos. Our results will contribute to extending the use of rabbit models for biomedical research.
DOI:10.1038/nature723URLPMID:11859353 [本文引用: 1]
Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.
DOI:10.1126/science.1086743URLPMID:12775846 [本文引用: 1]
DOI:10.1038/424635bURLPMID:12904779 [本文引用: 1]
Lithium-ion batteries are stabilized by an ultrathin protective film that is 10-50 nanometers thick and coats both electrodes. Here we artificially simulate the 'thermal-runaway' conditions that would arise should this coating be destroyed, which could happen in a battery large enough to overheat beyond 80 degrees C. We find that under these conditions the reaction of the battery electrolyte with the material of the unprotected positive electrode results in the formation of toxic fluoro-organic compounds. Although not a concern for the small units used in today's portable devices, this unexpected chemical hazard should be taken into account as larger and larger lithium-ion batteries are developed, for example for incorporation into electric-powered vehicles.
DOI:10.1126/science.1088313URLPMID:14512506 [本文引用: 2]
DOI:10.1038/436641aURLPMID:16079832 [本文引用: 1]
Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.
DOI:10.1095/biolreprod.109.081083URLPMID:19812298 [本文引用: 1]
In this study, we demonstrate the use of somatic cell nuclear transfer to produce the first cloned camelid, a dromedary camel (Camelus dromedarius) belonging to the family Camelidae. Donor karyoplasts were obtained from adult skin fibroblasts, cumulus cells, or fetal fibroblasts, and in vivo-matured oocytes, obtained from preovulatory follicles of superstimulated female camels by transvaginal ultrasound guided ovum pick-up, were used as cytoplasts. Reconstructed embryos were cultured in vitro for 7 days up to the hatching/hatched blastocyst stage before they were transferred to synchronized recipients on Day 6 after ovulation. Pregnancies were achieved from the embryos reconstructed from all cell types, and a healthy calf, named Injaz, was born from the pregnancy by an embryo reconstructed with cumulus cells. Genotype analyses, using 25 dromedary camel microsatellite markers, confirmed that the cloned calf was derived from the donor cell line and the ovarian tissue. In conclusion, the present study reports, for the first time, establishment of pregnancies and birth of the first cloned camelid, a dromedary camel (C. dromedarius), by use of somatic cell nuclear transfer. This has opened doors for the amelioration and preservation of genetically valuable animals like high milk producers, racing champions, and males of high genetic merit in camelids. We also demonstrated, for the first time, that adult and fetal fibroblasts can be cultured, expanded, and frozen without losing their ability to support the development of nuclear transfer embryos, a technology that may potentially be used to modify fibroblast genome by homologous recombination so as to generate genetically altered cloned animals.
DOI:10.1016/j.cell.2018.01.020URLPMID:29395327 [本文引用: 2]
Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2?healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42?surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.
DOI:10.1016/j.cell.2013.05.006URL [本文引用: 2]
Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.
DOI:10.1016/j.stem.2015.10.001URLPMID:26526725 [本文引用: 3]
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:10.1038/nature13287URL [本文引用: 2]
The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells(1-3), holding promise for autologous cell replacement therapy(4,5). Although methods to induce pluripotent stem cells from somatic cells by transcription factors(6) are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported(7-11), potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.
DOI:10.1126/science.1059399URLPMID:11326103 [本文引用: 1]
Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.
DOI:10.1016/s0092-8674(02)00681-5URLPMID:11955443 [本文引用: 1]
Immune-deficient Rag2(-/-) mice were used as nuclear donors for transfer into enucleated oocytes, and the resulting blastocysts were cultured to isolate an isogenic embryonic stem cell line. One of the mutated alleles in the Rag2(-/-) ES cells was repaired by homologous recombination, thereby restoring normal Rag2 gene structure. Mutant mice were treated with the repaired ES cells in two ways. (1) Immune-competent mice were generated from the repaired ES cells by tetraploid embryo complementation and were used as bone marrow donors for transplantation. (2) Hematopoietic precursors were derived by in vitro differentiation from the repaired ES cells and engrafted into mutant mice. Mature myeloid and lymphoid cells as well as immunoglobulins became detectable 3-4 weeks after transplantation. Our results establish a paradigm for the treatment of a genetic disorder by combining therapeutic cloning with gene therapy.
DOI:10.1038/nature06357URLPMID:18004281 [本文引用: 2]
Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.
DOI:10.1016/j.stem.2014.03.015URL [本文引用: 1]
Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.
DOI:10.1016/j.rbmo.2017.01.013URLPMID:28385334 [本文引用: 1]
Mutations in mitochondrial DNA (mtDNA) are maternally inherited and can cause fatal or debilitating mitochondrial disorders. The severity of clinical symptoms is often associated with the level of mtDNA mutation load or degree of heteroplasmy. Current clinical options to prevent transmission of mtDNA mutations to offspring are limited. Experimental spindle transfer in metaphase II oocytes, also called mitochondrial replacement therapy, is a novel technology for preventing mtDNA transmission from oocytes to pre-implantation embryos. Here, we report a female carrier of Leigh syndrome (mtDNA mutation 8993T > G), with a long history of multiple undiagnosed pregnancy losses and deaths of offspring as a result of this disease, who underwent IVF after reconstitution of her oocytes by spindle transfer into the cytoplasm of enucleated donor oocytes. A male euploid blastocyst wasobtained from the reconstituted oocytes, which had only a 5.7% mtDNA mutation load. Transfer of the embryo resulted in a pregnancy with delivery of a boy with neonatal mtDNA mutation load of 2.36-9.23% in his tested tissues. The boy is currently healthy at 7 months of age, although long-term follow-up of the child's longitudinal development remains crucial.
DOI:10.1089/153623003772032754URLPMID:14733743 [本文引用: 1]
DOI:10.1071/RD13329URLPMID:25482653 [本文引用: 3]
During the last 17 years, considerable advancements have been achieved in the production of pigs, transgenic and non-transgenic, by methods of somatic cell nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection, microinjection and sperm-mediated gene transfer by artificial insemination. Therefore, a review of the overall efficiency for the developmental competence of embryos produced by these in vitro methods would be useful in order to obtain a more thorough overview of this growing area with respect to its development and present status. In this review a meta-analysis was used to analyse data collected from all published articles with a focus on zygotes and embryos for transfer, pregnancy, full-term development and piglets born. It was generally concluded that an increasing level of in vitro manipulation of porcine embryos decreased the overall efficiency for production of piglets. The techniques of nuclear transfer have been developed markedly through the increasing number of studies performed, and the results have become more stable. Prolonged in vitro culture period did not lead to any negative effect on nuclear transfer embryos after their transfer and it resulted in a similar or even higher litter size. More complete information is needed in future scientific articles about these in vitro manipulation techniques to establish a more solid basis for the evaluation of their status and to reveal and further investigate any eventual problems.
DOI:10.1098/rstb.2011.0329URLPMID:23166393 [本文引用: 3]
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.
DOI:10.16288/j.yczz.15-466URLPMID:27232488 [本文引用: 1]
The cloning technique, also called somatic cell nuclear transfer (SCNT), has been successfully established and gradually applied to various mammalian species. However, the developmental rate of SCNT mammalian embryos is very low, usually at 1% to 5%, which limits the application of SCNT. Placental developmental defects are considered as the main cause of SCNT embryo development inhibition. Almost all of SCNT-derived mammalian placentas exhibit various abnormalities, such as placental hyperplasia, vascular defects and umbilical cord malformation. Mechanistically, these abnormalities result from failure of establishment of correct epigenetic modification in the trophectoderm genome, which leads to erroneous expression of important genes for placenta development-related, particularly imprinted genes. Consequently, aberrant imprinted gene expression gives rise to placental morphologic abnormalities and functional defects, therefore decreases developmental competence of cloned embryos. Currently, although numerous methods that can improve the developmental ability of SCNT-derived embryos have been reported, most of them are unable to substantially enhance the success rate of SCNT due to failure to eliminate the placental development defects. In this review, we summarize placental abnormalities and imprinted gene expression in mammalian cloning, and propose directions for the future research aiming to improve the cloning efficiency.
DOI:10.16288/j.yczz.15-466URLPMID:27232488 [本文引用: 1]
The cloning technique, also called somatic cell nuclear transfer (SCNT), has been successfully established and gradually applied to various mammalian species. However, the developmental rate of SCNT mammalian embryos is very low, usually at 1% to 5%, which limits the application of SCNT. Placental developmental defects are considered as the main cause of SCNT embryo development inhibition. Almost all of SCNT-derived mammalian placentas exhibit various abnormalities, such as placental hyperplasia, vascular defects and umbilical cord malformation. Mechanistically, these abnormalities result from failure of establishment of correct epigenetic modification in the trophectoderm genome, which leads to erroneous expression of important genes for placenta development-related, particularly imprinted genes. Consequently, aberrant imprinted gene expression gives rise to placental morphologic abnormalities and functional defects, therefore decreases developmental competence of cloned embryos. Currently, although numerous methods that can improve the developmental ability of SCNT-derived embryos have been reported, most of them are unable to substantially enhance the success rate of SCNT due to failure to eliminate the placental development defects. In this review, we summarize placental abnormalities and imprinted gene expression in mammalian cloning, and propose directions for the future research aiming to improve the cloning efficiency.
DOI:10.1016/j.tibtech.2016.03.008URLPMID:27118511 [本文引用: 2]
Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to 'rescue' endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs).
DOI:10.1016/j.placenta.2017.06.010URLPMID:28864025 [本文引用: 1]
Cloned piglets generated through somatic cell nuclear transfer (SCNT) have a high rate of neonatal death. Postnatal loss is associated with low birth weight, umbilical status and placental parameters in fertilisation-derived piglets. To investigate whether or not this relationship also exists in cloned piglets, birth weight, umbilical status, placental parameters, placental morphology and gene expression pattern were compared among four piglet groups, namely, SCNT-derived male piglets that died within 4 days (SCNT-DW4), SCNT-derived male piglets that survived over 4 days (SCNT-SO4), artificial insemination (AI)-generated male piglets that died within 4 days (AI-DW4) and AI-generated male piglets that survived over 4 days (AI-SO4). Results showed that the occurring frequency of abnormal umbilical cord in SCNT-DW4 piglets was significantly higher than that in AI-SO4 piglets but was similar to that in SCNT-SO4 and AI-DW4 piglets. The birth weight, placental surface area and placental weight of AI-SO4, AI-DW4 and SCNT-SO4 groups were similar but were significantly higher than those in SCNT-DW4 group. SCNT-SO4 placentas exhibited mild but SCNT-DW4 placentas showed severe morphological abnormalities compared with AI-SO4 placentas. The expression profiles of imprinting, angiopoiesis, nutrient transport, apoptosis and oxidative stress-related genes in SCNT-DW4 placentas were erroneous compared with those in SCNT-SO4 and AI-SO4 placentas, which both had similar gene expression patterns. These results indicate that birth weight, umbilical status, placental parameters, placental morphology and gene expression were associated with neonatal death of cloned piglets. The high loss of cloned piglets during neonatal age may be caused by severe deficiency of extra-embryonic development during prenatal stage.
DOI:10.1111/j.1439-0531.2008.01192.xURLPMID:18638154 [本文引用: 1]
Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.
DOI:10.1038/ng1973URLPMID:17325680 [本文引用: 4]
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:10.1016/j.cell.2006.02.024URLPMID:16530039 [本文引用: 1]
Sex chromosomes--particularly the human Y--have been a source of fascination for decades because of their unique transmission patterns and their peculiar cytology. The outpouring of genomic data confirms that their atypical structure and gene composition break the rules of genome organization, function, and evolution. The X has been shaped by dosage differences to have a biased gene content and to be subject to inactivation in females. The Y chromosome seems to be a product of a perverse evolutionary process that does not select the fittest Y, which may cause its degradation and ultimate extinction.
DOI:10.1016/j.tcb.2018.05.005URLPMID:29910081 [本文引用: 1]
In each somatic cell of a female mammal one X chromosome is transcriptionally silenced via X-chromosome inactivation (XCI), initiating early in development. Although XCI events are conserved in mouse and human postimplantation development, regulation of X-chromosome dosage in preimplantation development occurs differently. In preimplantation development, mouse embryos undergo imprinted form of XCI, yet humans lack imprinted XCI and instead regulate gene expression of both X chromosomes by dampening transcription. The long non-coding RNA Xist/XIST is expressed in mouse and human preimplantation and postimplantation development to orchestrate XCI, but its role in dampening is unclear. In this review, we discuss recent advances in our understanding of the role of Xist in X chromosome dosage compensation in mouse and human.
DOI:10.1002/wrna.1359URLPMID:27173581 [本文引用: 1]
X-chromosome inactivation (XCI) is a chromosome-wide regulatory process that ensures dosage compensation for X-linked genes in Theria. XCI is established during early embryogenesis and is developmentally regulated. Different XCI strategies exist in mammalian infraclasses and the regulation of this process varies also among closely related species. In Eutheria, initiation of XCI is orchestrated by a cis-acting locus, the X-inactivation center (Xic), which is particularly enriched in genes producing long noncoding RNAs (lncRNAs). Among these, Xist generates a master transcript that coats and propagates along the future inactive X-chromosome in cis, establishing X-chromosome wide transcriptional repression through interaction with several protein partners. Other lncRNAs also participate to the regulation of X-inactivation but the extent to which their function has been maintained in evolution is still poorly understood. In Metatheria, Xist is not conserved, but another, evolutionary independent lncRNA with similar properties, Rsx, has been identified, suggesting that lncRNA-mediated XCI represents an evolutionary advantage. Here, we review current knowledge on the interplay of X chromosome-encoded lncRNAs in ensuring proper establishment and maintenance of chromosome-wide silencing, and discuss the evolutionary implications of the emergence of species-specific lncRNAs in the control of XCI within Theria. WIREs RNA 2016, 7:702-722. doi: 10.1002/wrna.1359 For further resources related to this article, please visit the WIREs website.
DOI:10.1038/nature09457URLPMID:20962847 [本文引用: 1]
Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X?chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X?chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X?chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X?chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.
DOI:10.1016/j.semcdb.2016.04.014URLPMID:27112543 [本文引用: 1]
With the emergence of sex-determination by sex chromosomes, which differ in composition and number between males and females, appeared the need to equalize X-chromosomal gene dosage between the sexes. Mammals have devised the strategy of X-chromosome inactivation (XCI), in which one of the two X-chromosomes is rendered transcriptionally silent in females. In the mouse, the best-studied model organism with respect to XCI, this inactivation process occurs in different forms, imprinted and random, interspersed by periods of X-chromosome reactivation (XCR), which is needed to switch between the different modes of XCI. In this review, I describe the recent advances with respect to the developmental control of XCI and XCR and in particular their link to differentiation and pluripotency. Furthermore, I review the mechanisms, which influence the timing and choice, with which one of the two X-chromosomes is chosen for inactivation during random XCI. This has an impact on how females are mosaics with regard to which X-chromosome is active in different cells, which has implications on the severity of diseases caused by X-linked mutations.
DOI:10.1371/journal.pone.0010947URLPMID:20532033 [本文引用: 1]
Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.
DOI:10.1262/jrd.2016-095URLPMID:27569767 [本文引用: 3]
Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.
DOI:10.1126/science.1076997URLPMID:12351676 [本文引用: 1]
Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.
DOI:10.1101/sqb.2010.75.032URLPMID:21447823 [本文引用: 1]
Early development of female mammals is accompanied by transcriptional inactivation of one of their two X chromosomes. This leads to monoallelic expression of most of the X chromosome and ensures dosage compensation with respect to males (XY). One of the most surprising aspects of this phenomenon is that the two X homologs are treated differently even though they are present within the same nucleus. In eutherian mammals, such as humans and mice, either the maternal or the paternal X is inactivated during early embryogenesis. Once set up, the silent state is epigenetically transmitted as cells divide, so that adult females are mosaics of clonal cell populations, which express either of their two X chromosomes. The past years have been marked by the discovery of several molecular events that accompany chromosome-wide silencing.
DOI:10.1371/journal.pone.0011274URLPMID:20614022 [本文引用: 1]
Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos.
DOI:10.3390/ijms151221631URLPMID:25429426 [本文引用: 1]
Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.
DOI:10.1002/dvdy.24660URLPMID:30055068 [本文引用: 1]
Parthenogenetically activated oocytes exhibit poor embryo development and lower total numbers of cells per blastocyst accompanied by abnormally increased expression of Xist, a long noncoding RNA that plays an important role in triggering X chromosome inactivation during embryogenesis.
DOI:10.1002/mrd.22808URLPMID:28387970 [本文引用: 1]
XIST is an X-linked, non-coding gene responsible for the cis induction of X-chromosome inactivation (XCI). Knockout of the XIST allele on an active X chromosome abolishes erroneous XCI and enhances the in vivo development of cloned mouse embryos by more than 10-fold. This study aimed to investigate whether a similar manipulation would improve cloning efficiency in pigs. A male, porcine kidney cell line containing an EGFP insert in exon 1 of the XIST gene, resulting in a knockout allele (XIST-KO), was generated by homologous recombination using transcription activator-like effector nucleases (TALENs). The expression of X-linked genes in embryos cloned from the XIST-KO kidney cells was significantly higher than in male embryos cloned from wild-type (WT) kidney cells, but remained lower than that of in vivo fertilization-produced counterparts. The XIST-KO cloned embryos also had a significantly lower blastocyst rate and a reduced full-term development rate compared to cloned WT embryos. These data suggested that while mutation of a XIST gene can partially rescue abnormal XCI, it cannot improve the developmental efficiency of cloned male porcine embryos-a deficiency that may be caused by incomplete rescue of abnormal XCI and/or by long-term drug selection of the XIST-KO nuclear donor cells, which might adversely affect the developmental efficiency of embryos created from them.
DOI:10.1016/j.stemcr.2017.12.015URLPMID:29337117 [本文引用: 3]
Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in?vivo long-term developmental capacity of porcine NT embryos.
URLPMID:17951162 [本文引用: 1]
DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide) while CpG islands located in gene promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting. The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis, may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA. Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called "non-CpG" methylation). The knowledge of a normal methylation process and its aberrations appeared to be useful while searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique (using primers for methylated and unmethylated sequences) five new genes which are potential biomarkers of lung cancer have been presented.
DOI:10.3724/SP.J.1005.2010.00762URL [本文引用: 1]
体细胞核移植在农业应用、生产疾病模型动物、转基因家畜或产生人胚胎干细胞来治疗人类的疾病方面有巨大的应用潜力。虽然已经成功克隆出多种哺乳动物, 但该技术仍存在一些未解决的问题, 包括产生克隆动物的效率低和克隆动物的异常等。异常的表观遗传重编程是克隆胚胎发育失败的一个重要因素。文章重点论述了DNA甲基化、组蛋白修饰及其与克隆胚胎发育的关系。了解表观遗传调控机制有助于解决核移植技术中存在的问题, 有利于更好地应用这项技术。
DOI:10.3724/SP.J.1005.2010.00762URL [本文引用: 1]
体细胞核移植在农业应用、生产疾病模型动物、转基因家畜或产生人胚胎干细胞来治疗人类的疾病方面有巨大的应用潜力。虽然已经成功克隆出多种哺乳动物, 但该技术仍存在一些未解决的问题, 包括产生克隆动物的效率低和克隆动物的异常等。异常的表观遗传重编程是克隆胚胎发育失败的一个重要因素。文章重点论述了DNA甲基化、组蛋白修饰及其与克隆胚胎发育的关系。了解表观遗传调控机制有助于解决核移植技术中存在的问题, 有利于更好地应用这项技术。
[本文引用: 1]
[本文引用: 1]
DOI:10.3724/SP.J.1005.2014.0403URL [本文引用: 1]
DNA甲基化通过调节基因转录、印记、X染色体灭活和防御外源性遗传物质入侵等, 在细胞分化、胚胎发育、环境适应和疾病发生发展上发挥重要作用, 是当前表观遗传学研究的热点领域之一。文章介绍了在过去几年中TET介导的DNA羟甲基化及其在早期胚胎发育中的作用, DNA主动去甲基化及其与被动去甲基化的关系, DNA甲基化建立及其与组蛋白修饰、染色质构象、多梳蛋白和非编码RNA结合等关系方面的重要研究进展和存在的问题以及DNA甲基化的转化应用前景。
DOI:10.3724/SP.J.1005.2014.0403URL [本文引用: 1]
DNA甲基化通过调节基因转录、印记、X染色体灭活和防御外源性遗传物质入侵等, 在细胞分化、胚胎发育、环境适应和疾病发生发展上发挥重要作用, 是当前表观遗传学研究的热点领域之一。文章介绍了在过去几年中TET介导的DNA羟甲基化及其在早期胚胎发育中的作用, DNA主动去甲基化及其与被动去甲基化的关系, DNA甲基化建立及其与组蛋白修饰、染色质构象、多梳蛋白和非编码RNA结合等关系方面的重要研究进展和存在的问题以及DNA甲基化的转化应用前景。
DOI:10.1038/nrg.2017.33URLPMID:28555658 [本文引用: 1]
In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodified cytosine (C) through TET dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair. In the past few years, biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA demethylation. Additionally, many regulatory mechanisms of this process have been identified. Technological advances in mapping and tracing the oxidized forms of 5mC allow further dissection of their functions. Furthermore, the biological functions of active DNA demethylation in various biological contexts have also been revealed. In this Review, we summarize the recent advances and highlight key unanswered questions.
DOI:10.1038/35000656URLPMID:10676950 [本文引用: 1]
DOI:10.1038/ncb1176URLPMID:15448701 [本文引用: 1]
Nuclear transplantation experiments in amphibia and mammals have shown that oocyte and egg cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression and new pathways of cell differentiation; however, very little is known about the molecular mechanism of nuclear reprogramming. Here we have used nuclear and DNA transfer from mammalian somatic cells to analyse the mechanism of activation of the stem cell marker gene oct4 by Xenopus oocytes. We find that the removal of nuclear protein accelerates the rate of reprogramming, but even more important is the demethylation of somatic cell DNA. DNA demethylation seems to precede gene reprogramming, and is absolutely necessary for oct4 transcription. Reprogramming by oocytes occurs in the absence of DNA replication and RNA/protein synthesis. It is also selective, operating only on the promoter, but not enhancers, of oct4; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and transcription. We conclude that the demethylation of promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei.
DOI:10.7554/eLife.40757URLPMID:30468428 [本文引用: 1]
DNA methylation plays an essential role in mammalian genomes and expression of the responsible enzymes is tightly controlled. Deregulation of the de novo DNA methyltransferase DNMT3B is frequently observed across cancer types, yet little is known about its ectopic genomic targets. Here, we used an inducible transgenic mouse model to delineate rules for abnormal DNMT3B targeting, as well as the constraints of its activity across different cell types. Our results explain the preferential susceptibility of certain CpG islands to aberrant methylation and point to transcriptional state and the associated chromatin landscape as the strongest predictors. Although DNA methylation and H3K27me3 are usually non-overlapping at CpG islands, H3K27me3 can transiently co-occur with DNMT3B-induced DNA methylation. Our genome-wide data combined with ultra-deep locus-specific bisulfite sequencing suggest a distributive activity of ectopically expressed Dnmt3b that leads to discordant CpG island hypermethylation and provides new insights for interpreting the cancer methylome.
DOI:10.1126/science.1212483URLPMID:21940858 [本文引用: 1]
Although global erasure of DNA methylation has been observed in zygotes and primordial germ cells, the responsible enzyme(s) have been elusive. The demonstration that members of the Tet (ten eleven translocation) family of proteins are capable of catalyzing conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC) raises the possibility that Tet proteins may participate in this process. Indeed, recent studies have implicated the involvement of Tet3 in the conversion of 5mC to 5hmC in zygotes. This result, combined with the demonstration that Tet proteins can further oxidize 5hmC to 5-carboxylcytosine followed by excision by thymine-DNA glycosylase, raises the possibility that active demethylation may take place in a process that involves Tet3-mediated oxidation followed by base excision repair. We demonstrated by immunostaining of mitotic chromosome spreads of preimplantation embryos that the 5hmC associated with the paternal genome in zygotes is gradually lost during preimplantation development. Our study suggests that, although the conversion of 5mC to 5hmC in zygotes is an enzyme-catalyzed process, loss of 5hmC during preimplantation appears to be a DNA replication-dependent passive process.
[本文引用: 4]
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:10.1038/nrd772URLPMID:12120280 [本文引用: 2]
The opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) allow gene expression to be exquisitely regulated through chromatin remodelling. Aberrant transcription due to altered expression or mutation of genes that encode HATs, HDACs or their binding partners, is a key event in the onset and progression of cancer. HDAC inhibitors can reactivate gene expression and inhibit the growth and survival of tumour cells. The remarkable tumour specificity of these compounds, and their potency in vitro and in vivo, underscore the potential of HDAC inhibitors as exciting new agents for the treatment of cancer.
DOI:10.3724/SP.J.1005.2014.1211URL [本文引用: 1]
体细胞核移植(Somatic cell nuclear transfer, SCNT)是指将高度分化的体细胞移入到去核的卵母细胞中发育并最终产生后代的技术。然而, 体细胞克隆的总体效率仍然处于一个较低的水平, 主要原因之一是由于体细胞供体核不完全的表观遗传重编程, 包括DNA甲基化、组蛋白乙酰化、基因组印记、X染色体失活和端粒长度等修饰出现的异常。使用一些小分子化合物以及Xist基因的敲除或敲低等方法能修复表观遗传修饰错误, 辅助供体核的重编程, 从而提高体细胞克隆效率, 使其更好地应用于基础研究和生产实践。文章对体细胞核移植后胚胎发育过程中出现的异常表观遗传修饰进行了综述, 并着重论述了近年来有关修复表观遗传错误的研究进展。
DOI:10.3724/SP.J.1005.2014.1211URL [本文引用: 1]
体细胞核移植(Somatic cell nuclear transfer, SCNT)是指将高度分化的体细胞移入到去核的卵母细胞中发育并最终产生后代的技术。然而, 体细胞克隆的总体效率仍然处于一个较低的水平, 主要原因之一是由于体细胞供体核不完全的表观遗传重编程, 包括DNA甲基化、组蛋白乙酰化、基因组印记、X染色体失活和端粒长度等修饰出现的异常。使用一些小分子化合物以及Xist基因的敲除或敲低等方法能修复表观遗传修饰错误, 辅助供体核的重编程, 从而提高体细胞克隆效率, 使其更好地应用于基础研究和生产实践。文章对体细胞核移植后胚胎发育过程中出现的异常表观遗传修饰进行了综述, 并着重论述了近年来有关修复表观遗传错误的研究进展。
DOI:10.1016/j.bbrc.2005.11.164URLPMID:16356478 [本文引用: 1]
The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.
DOI:10.1095/biolreprod.105.047456URLPMID:16481594 [本文引用: 1]
Before fertilization, chromatins of both mouse oocytes and spermatozoa contain very few acetylated histones. Soon after fertilization, chromatins of both gametes become highly acetylated. The same deacetylation-reacetylation changes occur with histones of somatic nuclei transferred into enucleated oocytes. The significance of these events in somatic chromatin reprogramming to the totipotent state is not known. To investigate their importance in reprogramming, we injected cumulus cell nuclei into enucleated mouse oocytes and estimated the histone deacetylation dynamics with immunocytochemistry. Other reconstructed oocytes were cultured before and/or after activation in the presence of the highly potent histone deacetylase inhibitor trychostatin A (TSA) for up to 9 h postactivation. The potential of TSA-treated and untreated oocytes to develop to the blastocyst stage and to full term was compared. Global deacetylation of histones in the cumulus nuclei occurred between 1 and 3 h after injection. TSA inhibition of histone deacetylation did not affect the blastocyst rate (37% with and 34% without TSA treatment), whereas extension of the TSA treatment beyond the activation point significantly increased the blastocyst rate (up to 81% versus 40% without TSA treatment) and quality (on average, 59 versus 45 cells in day 4 blastocysts with and without TSA treatment, respectively). TSA treatment also slightly increased full-term development (from 0.8% to 2.8%). Thus, deacetylation of somatic histones is not important for reprogramming, and hyperacetylation might actually improve reprogramming.
DOI:10.1038/srep10127URLPMID:25974394 [本文引用: 2]
Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage.
DOI:10.4161/cc.29215URL [本文引用: 2]
Recent studies have shown that DNA damage affects embryo development and also somatic cell reprogramming into induced pluripotent stem (iPS) cells. It has been also shown that treatment with histone deacetylase inhibitors (HDACi) improves development of embryos produced by somatic cell nuclear transfer (SCNT) and enhances somatic cell reprogramming. There is evidence that increasing histone acetylation at the sites of DNA double-strand breaks (DSBs) is critical for DNA damage repair. Therefore, we hypothesized that HDACi treatment enhances cell programming and embryo development by facilitating DNA damage repair. To test this hypothesis, we first established a DNA damage model wherein exposure of nuclear donor cells to ultraviolet (UV) light prior to nuclear transfer reduced the development of SCNT embryos proportional to the length of UV exposure. Detection of phosphorylated histone H2A.x (H2AX139ph) foci confirmed that exposure of nuclear donor cells to UV light for 10 s was sufficient to increase DSBs in SCNT embryos. Treatment with HDACi during embryo culture increased development and reduced DSBs in SCNT embryos produced from UV-treated cells. Transcript abundance of genes involved in either the homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways for DSBs repair was reduced by HDACi treatment in developing embryos at day 5 after SCNT. Interestingly, expression of HR and NHEJ genes was similar between HDACi-treated and control SCNT embryos that developed to the blastocyst stage. This suggested that the increased number of embryos that could achieve the blastocyst stage in response to HDACi treatment have repaired DNA damage. These results demonstrate that DNA damage in nuclear donor cells is an important component affecting development of SCNT embryos, and that HDACi treatment after nuclear transfer enhances DSBs repair and development of SCNT embryos.
DOI:10.1016/j.bbrc.2014.11.051URLPMID:25446119 [本文引用: 1]
In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P<0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P<0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.
DOI:10.1262/jrd.10-058aURLPMID:20962457 [本文引用: 1]
We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines.
DOI:10.1007/s11626-018-0272-4URLPMID:29943354 [本文引用: 1]
SCNT technology has been successfully used to clone a variety of mammals, but the cloning efficiency is very low. This low efficiency is likely due to the incomplete reprogramming of SCNT embryos. Histone modification and DNA methylation may participate in these events. Thus, it would be interesting to attempt to improve the efficiency of SCNT by using a HDACi VPA. In order to guarantee the effect of VPA and reduce its cytotoxicity, a comprehensive analysis of the cell proliferation and histone modification was performed. The results showed that 0.5 and 1?mM VPA treatment for 24?h were the optimal condition. According to the results, H3K4me3 was increased in 0.5 and 1?mM VPA groups, whereas H3K9me2 was significantly decreased. These are the signals of gene-activation. In addition, VPA treatment led to the overexpression of Oct4 and Nanog. These indicated that VPA-treated cells had similar patterns of histone to zygotic embryos, and may be more favorable for reprograming. A total of 833 cloned embryos were produced from the experimental replicates of VPA-treated donor cells. In 1?mM treatment group, the blastocyst rates were significantly increased compared with control. At the same time, our findings demonstrated the interrelation between DNA methylation and histone modifications.
DOI:10.1071/RD12336URLPMID:23506644 [本文引用: 1]
Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3mM VPA for 24h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage.
DOI:10.1089/cell.2009.0038URLPMID:20132015 [本文引用: 1]
Faulty epigenetic reprogramming of somatic nuclei is likely to be a major cause of low success observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been demonstrated that the developmental competence of SCNT embryos in several species were significantly enhanced via treatment of histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) to increase histone acetylation. Here we report that 50 nM TSA for 10 h after activation increased the developmental competence of porcine SCNT embryos constructed from Landrace fetal fibroblast cells (FFCs) in vitro and in vivo, but not at higher concentrations. Therefore, we optimized the application of another novel HDACi, Scriptaid, for development of porcine SCNT embryos. We found that treatment with 500 nM Scriptaid significantly enhanced the development SCNT embryos to the blastocyst stage when outbred Landrace FFCs and ear fibroblast cells (EFCs) were used as donors compared to the untreated group. Scriptaid increased the overall cloning efficiency from 0.4% (untreated group) to 1.6% for Landrace FFCs and 0 to 3.7% for Landrace EFCs. Moreover, treatment of SCNT embryos with Scriptaid improved the histone acetylation on Histone H4 at lysine 8 (AcH4K8) in a pattern similar to that of the in vitro fertilized (IVF) embryos.
DOI:10.1089/cell.2009.0047URLPMID:20132016 [本文引用: 1]
The present study assessed changes in epigenetic markers and pre- and postimplantation development in somatic cell nuclear transfer (SCNT) porcine embryos after treatment with the inhibitor of histone deacetylases (HDACi), Trichostatin A (TSA). Embryos were generated using in vitro matured oocytes and nuclei from either a male fetal fibroblast (FF) cell line or bone marrow cells (BMC) from three adult sows. After nuclear transfer, oocytes were either exposed or not to 10 ng/mL TSA for 10 h starting 1 h after cell fusion. Samples of one-cell stage and cleaved (two- to four-cell stage) embryos were fixed at 15 to 18 h or 46 to 48 h after cell fusion and immunocytochemically processed to detect histone H3 acetylation at lysine 14 (H3K14ac) or histone H3 dimethylation at lysine 9 (H3K9m2) using specific primary antibodies. TSA treatment increased the immunofluorescent signal for H3K14ac in cleaved embryos derived from both FF and BMC but did not affect H3K9m2. Development to the blastocyst stage was increased by TSA treatment (45.2 vs. 23.9%) in embryos produced from FF cells but not in those produced from BMC (30.6 vs. 27.4%). Cloned piglets were produced from both treatments when day 5 to 6 blastocyst-stage embryos derived from FF cells were transferred into the uterus of recipient females. Cloned piglets were also produced after the transfer of TSA-treated blastocysts derived from BMC of adult sows but not from control embryos. These findings suggest that the inhibition of histone deacetylases have similar effects on enhancing H3K14ac in SCNT embryos reconstructed from different cell types but the effect on in vitro and in vivo development seems to differ according to the nuclear donor cell type.
DOI:10.1038/s41556-018-0093-4URLPMID:29686265 [本文引用: 1]
H3K9me3-dependent heterochromatin is a major barrier of cell fate changes that must be reprogrammed after fertilization. However, the molecular details of these events are lacking in early embryos. Here, we map the genome-wide distribution of H3K9me3 modifications in mouse early embryos. We find that H3K9me3 exhibits distinct dynamic features in promoters and long terminal repeats (LTRs). Both parental genomes undergo large-scale H3K9me3 reestablishment after fertilization, and the imbalance in parental H3K9me3 signals lasts until blastocyst. The rebuilding of H3K9me3 on LTRs is involved in silencing their active transcription triggered by DNA demethylation. We identify that Chaf1a is essential for the establishment of H3K9me3 on LTRs and subsequent transcriptional repression. Finally, we find that lineage-specific H3K9me3 is established in post-implantation embryos. In summary, our data demonstrate that H3K9me3-dependent heterochromatin undergoes dramatic reprogramming during early embryonic development and provide valuable resources for further exploration of the epigenetic mechanism in early embryos.
DOI:10.1016/j.celrep.2018.04.036URLPMID:29768195 [本文引用: 1]
Mammalian oocytes have the ability to reset the transcriptional program of differentiated somatic cells into that of totipotent embryos through somatic cell nuclear transfer (SCNT). However, the mechanisms underlying SCNT-mediated reprogramming are largely unknown. To understand the mechanisms governing chromatin reprogramming during SCNT, we profiled DNase I hypersensitive sites (DHSs) in donor cumulus cells and one-cell stage SCNT embryos. To our surprise, the chromatin accessibility landscape of the donor cells is drastically changed to recapitulate that of the in?vitro fertilization (IVF)-derived zygotes within 12?hr. Interestingly, this DHS reprogramming takes place even in the presence of a DNA replication inhibitor, suggesting that SCNT-mediated DHS reprogramming is independent of DNA replication. Thus, this study not only reveals the rapid and drastic nature of the changes in chromatin accessibility through SCNT but also establishes a DNA replication-independent model for studying cellular reprogramming.
DOI:10.1242/dev.158261URLPMID:29453221 [本文引用: 1]
Aberrant epigenetic reprogramming often results in developmental defects in somatic cell nuclear transfer (SCNT) embryos during embryonic genome activation (EGA). Bovine eight-cell SCNT embryos exhibit global hypermethylation of histone H3 lysine 9 tri- and di-methylation (H3K9me3/2), but the intrinsic reason for this remains elusive. Here, we provide evidence that two H3K9 demethylase genes, lysine-specific demethylase 4D (KDM4D) and 4E (KDM4E), are related to active H3K9me3/2 demethylation in in vitro fertilized (IVF) embryos and are deficiently expressed in cloned embryos at the time of EGA. Moreover, KDM4E plays a more crucial role in IVF and SCNT embryonic development, and overexpression of KDM4E can restore the global transcriptome, improve blastocyst formation and increase the cloning efficiency of SCNT embryos. Our results thereby indicate that KDM4E can function as a crucial epigenetic regulator of EGA and as an internal defective factor responsible for persistent H3K9me3/2 barriers to SCNT-mediated reprogramming. Furthermore, we show that interactions between RNA and KDM4E are essential for H3K9 demethylation during EGA. These observations advance the understanding of incomplete nuclear reprogramming and are of great importance for transgenic cattle procreation.
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DOI:10.1038/nature23262URLPMID:28723896 [本文引用: 1]
Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashions. After fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci, including imprinting control regions. How parental chromatin becomes equalized and how imprinting control regions escape from this reprogramming is largely unknown. Here we profile parental allele-specific DNase I hypersensitive sites in mouse zygotes and morula embryos, and investigate the epigenetic mechanisms underlying these allelic sites. Integrated analyses of DNA methylome and tri-methylation at lysine 27 of histone H3 (H3K27me3) chromatin immunoprecipitation followed by sequencing identify 76 genes with paternal allele-specific DNase I hypersensitive sites that are devoid of DNA methylation but harbour maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induces maternal allele expression. H3K27me3-dependent imprinting is largely lost in the embryonic cell lineage, but at least five genes maintain their imprinted expression in the extra-embryonic cell lineage. The five genes include all paternally expressed autosomal imprinted genes previously demonstrated to be independent of oocyte DNA methylation. Thus, our study identifies maternal H3K27me3 as a DNA methylation-independent imprinting mechanism.
DOI:10.1101/gad.304113.117URLPMID:29089420 [本文引用: 2]
Maternal imprinting at the Xist gene is essential to achieve paternal allele-specific imprinted X-chromosome inactivation (XCI) in female mammals. However, the mechanism underlying Xist imprinting is unclear. Here we show that the Xist locus is coated with a broad H3K27me3 domain that is established during oocyte growth and persists through preimplantation development in mice. Loss of maternal H3K27me3 induces maternal Xist expression and maternal XCI in preimplantation embryos. Our study thus identifies maternal H3K27me3 as the imprinting mark of Xist.
DOI:10.1016/j.theriogenology.2007.08.033URLPMID:17945341 [本文引用: 1]
Somatic cell nuclear transfer (SCNT) has been accomplished in an ever-growing list of species. In each case, an enucleated oocyte has successfully reset the nucleus of a somatic cell such that the embryonic program could progress to the production of a live offspring. The overall efficiency of the process remains low due to a combination of biological and technical challenges, some of which are known and others remain to be elucidated. Comparative studies between livestock and laboratory species may help improve not only nuclear transfer efficiencies but also uncover basic underlying developmental principles.
DOI:10.1017/S0967199417000673URLPMID:29239295 [本文引用: 1]
Somatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.
DOI:10.16288/j.yczz.18-209URLPMID:30369467 [本文引用: 1]
The research of stem cell field has developed rapidly in recent decades. With the enhancing of China's economic power, the strength of scientific research is steadily increasing, and the stem cell research even has reached international advanced level. Here, we make a historical review of important research in China, focusing on somatic cell nuclear transfer, induced pluripotent stem cells, haploid pluripotent stem cells and early embryo development. The fast development of single cell sequencing will greatly help the understanding of various development process, and the stem cell clinical application will blossom in China in the future.
DOI:10.16288/j.yczz.18-209URLPMID:30369467 [本文引用: 1]
The research of stem cell field has developed rapidly in recent decades. With the enhancing of China's economic power, the strength of scientific research is steadily increasing, and the stem cell research even has reached international advanced level. Here, we make a historical review of important research in China, focusing on somatic cell nuclear transfer, induced pluripotent stem cells, haploid pluripotent stem cells and early embryo development. The fast development of single cell sequencing will greatly help the understanding of various development process, and the stem cell clinical application will blossom in China in the future.
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.
DOI:10.1016/j.cell.2017.06.029URLPMID:28709003 [本文引用: 1]
High-order chromatin structure plays important roles in gene expression regulation. Knowledge of the dynamics of 3D chromatin structures during mammalian embryo development remains limited. We report the 3D chromatin architecture of mouse gametes and early embryos using an optimized Hi-C method with low-cell samples. We find that mature oocytes at the metaphase II stage do not have topologically associated domains (TADs). In sperm, extra-long-range interactions (>4 Mb) and interchromosomal interactions occur frequently. The high-order structures of both the paternal and maternal genomes in zygotes and two-cell embryos are obscure but are gradually re-established through development. The establishment of the TAD structure requires DNA replication but not zygotic genome activation. Furthermore, unmethylated CpGs are enriched in A compartment, and methylation levels are decreased to a greater extent in A compartment than in B compartment in embryos. In summary, the global reprogramming of chromatin architecture occurs during early mammalian development.
DOI:10.1038/nature15749URLPMID:26659182 [本文引用: 1]
Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
DOI:10.1016/j.cell.2012.09.045URL [本文引用: 1]
The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hr of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include an unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed.
DOI:10.1038/ncb2768URLPMID:23748610 [本文引用: 1]
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.
DOI:10.1093/hmg/ddt495URL [本文引用: 1]
Animals cloned by somatic cell nuclear transfer (SCNT) provide a unique model for understanding the mechanisms of nuclear epigenetic reprograming to a state of totipotency. Though many phenotypic abnormalities have been demonstrated in cloned animals, the underlying mechanisms are not well understood. In this study, we performed transcriptome-wide allelic expression analyses in brain and placental tissues of cloned mice. We found that Gab1, Sfmbt2 and Slc38a4 showed loss of imprinting in all cloned mice analyzed, which might be involved in placentomegaly of cloned mice. These three genes did not require de novo DNA methylation in growing oocytes for the establishment of imprinting, implying the involvement of a de novo DNA methylation-independent mechanism. Loss of Dlk1-Dio3 imprinting was also observed in nearly half of cloned mouse embryos and showed a strong correlation with embryonic lethality. Our findings are essential to understand the underlying mechanisms of developmental abnormalities of cloned animals. We also emphasize that particular attention should be paid to specific imprinted genes for therapeutic and agricultural applications of SCNT.
DOI:10.1016/j.cell.2019.01.006URLPMID:30712874 [本文引用: 1]
Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the?regulatory programs that underlie them. We apply?the method to reconstruct the landscape of?reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18?days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate?that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.
DOI:10.1007/978-1-4939-8831-0_22URLPMID:30353526 [本文引用: 1]
The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of pooled CRISPR/Cas9 targeting vectors.
DOI:10.1038/nbt.2842URLPMID:24584096 [本文引用: 1]
Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
