体细胞核移植后表观遗传重编程的异常及其修复

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。     

家畜体细胞克隆中核重编程机理研究进展

体细胞克隆在绵羊、山羊、牛、猪等家畜中获得了成功,但目前的克隆效率非常低。克隆效率低使家畜体细胞克隆技术在畜牧业生产及其他领域的应用受到极大的限制,问题的根源在于对体细胞克隆中核重编程的分子机理缺乏了解。供体细胞核移入去核的卵母细胞后,必须经过后成表观遗传修饰的重编程,从而恢复供体细胞核的全能性,才能保证重构胚的正常发育及个体的正常生长。本文从移植核的重构、DNA甲基化总体改变、组蛋白修饰、X染色体失活、端粒长度和端粒酶活性恢复、印迹基因及其他与发育相关基因的表达及核重编程的影响因素等几个方面探讨了体细胞克隆中的核重编程机理,为克隆效率提高的方法研究提供理论依据。     

完整的供体细胞膜及供体胞质对小鼠体细胞克隆胚发育的影响

利用显微注射和电融合的方法都可以成功地获得体细胞克隆小鼠, 由于电融合法操作耗时, 融合率低, 因而大多数克隆小鼠是采用注射方法。而注射法需要将供体细胞核从细胞中分离出来, 此分离操作有可能导致对DNA的损伤, 曾有人使用直径较粗的注射管进行完整的供体细胞注射, 这种方法操作相对简单而且对供体核没有损伤。为了研究这种方法在小鼠核移植中是否适用, 本实验使用完整的小鼠卵丘细胞作供体, 进行显微注射, 结果显示, 完整的卵丘细胞注入卵母细胞后, 无论在1小时或者6小时激活, 大部分的重构胚在2细胞期碎裂, 而去掉细胞膜的供体体细胞核注入卵母细胞后, 重构胚可以卵裂并进一步发育。卵母细胞去核后不注射供体也发生碎裂, 大部分的孤雌胚(不去核)在完整的卵丘细胞被注入后同样发生碎裂。在供体卵丘细胞刚破膜后即被注入卵胞质和供核被充分剥离后注入两种情况下获得的重构胚的体外发育中, 前者发育各期的比率显著低于后者。这些结果说明完整的卵丘细胞膜阻碍了卵胞质对体细胞核的重编程作用, 造成碎裂; 注入卵胞质的供体质膜和胞质成分影响了克隆胚的体外发育。     

母源物质对重编程作用研究进展

卵母细胞发生过程中会积累大量的物质,即所谓的母源物质(maternal materials),自然状态下,这些母源物质对受精以及之后的发育具有重要的生物学功能。雌雄配子融合后,精子核与卵母细胞中单倍体染色体组均会发生剧烈的表观遗传修饰变化,这个过程也同样发生在体细胞核移植到卵母细胞质之后,这种变化被称之为重编程。重编程奠定了新个体发生发育全部程序的基础,因此是一个备受重视的生物学过程。重编程包括DNA去甲基化、染色质重塑和组蛋白修饰等。受精后,卵母细胞与精子的基因组均会在一定时间和空间范围内经历相应的重编程过程,清除各自基因组在配子形成中保留的表观遗传学修饰,调控基因表达并形成正常发育的全能性胚胎。受精后,卵母细胞成熟中积累的多种母源物质聚集在雄原核周围,调控其基因组的重编程。体细胞核移植胚胎中供体细胞核注到去核卵母细胞后也将在卵母细胞中蛋白质、mRNA、酶类等母源物质的作用下进行重编程。现总结了母源物质对雄原核及供体细胞核重编程作用的研究进展,并探讨了母源物质作用的可能机制。     

哺乳动物体细胞异种核移植胚胎早期发育的研究进展

体细胞异种核移植是指将一个物种的体细胞移植到另一物种的去核卵母细胞中,移入的体细胞核在受体胞质中重编程并发育成新个体的实验方法.该方法为拯救濒危物种和获取灵长类胚胎干细胞提供了可能的途径.但这方面的研究目前还只获得初步的进展,核重编程不完全以及异种胚胎的囊胚率低仍是其面临的主要难点.本文从基因表达、表观重编程、线粒体异质性、核重塑和核移植体系优化等方面入手,介绍近年来哺乳动物体细胞异种核移植的研究进展,并探讨异种重构胚重编程所面临的关键问题和可能获得成功的方法.     

哺乳动物克隆的原理与途径

在近年来哺乳动物克隆技术研究的基础上,阐述了克隆的定义及其原理。叙述了作为核供体与受体细胞周期的协调性问题,列出了使供体细胞处于特定细胞周期的一些处理方法。同时也叙述了针对不同时期的供体核对受体卵母细胞采取的不同处理。简述了不同类型的显微操作、去核、电融合及化学激活等方法,提出了哺乳动物克隆所面临的一些重要问题。     

人-山羊异种核移植胚胎发育的初步研究

以体外分离培养的人胚胎成纤维细胞为核供体,经血清饥饿培养后,通过显微操作技术移入山羊去核卵母细胞中,采用化学方法激活重组胚.通过体外培养观察,2-细胞胚胎发育率可达51.33%,4-细胞发育率为31.42%,但发育至桑椹胚阶段的胚胎数目大大减少,仅为9.73%.虽然目前尚未能获得异种核移植囊胚,但实验结果说明山羊成熟卵母细胞可以支持人体细胞核完成重编程,人-山羊异种体细胞核移植重组胚可在体外完成其早期发育.     

体细胞核移植诱导的表观遗传学变化

体细胞核移植技术是指将一个分化的体细胞核置入去核的卵母细胞中,并发育产生与供体细胞遗传背景一致的克隆后代的技术。目前,世界上通过体细胞核移植技术已经产生了许多的克隆动物。但克隆过程中还存在着很多问题,比如,克隆效率太低、克隆个体常伴有表型异常和早亡等,从而使该技术应有的应用潜力不能得到充分的发挥。体细胞表观遗传学重编程的不完全或紊乱是造成核移植诸多问题的主要原因。近十多年来,人们对体细胞核移植后的重编程进行了广泛的研究,其核心内容包括核及核外结构的重塑、DNA甲基化模式的重建、基因印迹和x染色体失活、组蛋白乙酰化模式的重建、端粒长度恢复等,以期能够对其重编程加以人为干预,从而提高动物克隆效率。本文拟对体细胞核移植诱导的重编程研究进展加以综述,希望对体细胞重编程机制的阐明有所启发。     

动物克隆及其相关重编程机制研究

体细胞核移植技术已成功克隆出绵羊、牛、小鼠、猪、猕猴等多种动物,克隆技术也被广泛应用于畜牧业、生物医学、基础科研等众多领域。但是克隆的成功率较低,且克隆后代经常出现各种畸形,关键原因之一就是供体细胞重编程不完全。供体细胞核在进入去核卵后,会经历核膜降解、早熟染色质凝集、卵母细胞激活、核扩张、合子基因组激活等一系列事件,期间会发生染色质结构重编程、组蛋白变体合并、组蛋白修饰重编程、DNA甲基化重编程等多种重编程过程,只有重编程成功的胚胎才能正常发育成个体。该文总结了近年来克隆中重编程研究的进展并介绍了新兴的半克隆技术,希望以此加深对重编程机制的了解,从而使克隆的效率得到提高。     

体细胞核移植后核重编程的影响因素

近年来,人类核移植胚胎干细胞建系成为一项炙手可热的研究,用再生医学的理念治疗退行性疾病及器官移植为这一研究带来无穷的魅力和生命力;但是核重编程仍是核移植技术的瓶颈,制约了重构胚胎干细胞的研究。核重编程是指供体细胞核移入卵母细胞后必须停止本身的基因表达程序并恢复为胚胎发育所必需的特定的胚胎表达程序。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核的表遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。一般来说,颗粒细胞作为核供体最易被核重编程。供核者为胎体或新生体,供核细胞处于低分化状态或已传数代,供核细胞经过去表遗传标记处理,供卵者性成熟且年龄轻、卵子核与胞浆都成熟等均为有利于核重编程的因素。重构胚胎的培养方法对核重编程也至关重要,目前主张使用序贯培养及体细胞化培养。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞研究,提高重构胚胎干细胞建系效率。     

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.     

Nuclear cloning,stem cells,and genomic reprogramming

The generation of adult animals by nuclear cloning from adult donor cells is extremely inefficient, with most clones dying soon after implantation. In contrast, cloning from embryonic stem cell donor nuclei is significanty more efficient than from adult donor cells. However, regardless of donor cell type, all clones that survive to birth and beyond suffer serious phenotypic and gene expression abnormalities. All available evidence is consistent with the notion that the anomalous phenotypes of cloned animals are caused by faulty epigenetic reprogramming of the donor nucleus. Faulty reprogramming appears to be caused by the cloning process itself as well as by the epigenetic state of the donor nucleus. In contrast to reproductive cloning, faulty reprogramming of the donor nucleus does not tend to interfere with the application of nuclear transfer technology for therapeutic purposes (therapeutic cloning).     

Nuclear transplantation in the pig embryo: nuclear swelling

The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase II oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4-, 8-, and 16-cell stage embryos swelled to a similar diameter after nuclear transfer (26.9, 27.3, and 27.2 microns, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3, and 13.0 microns, respectively). This is a swelling of 47, 91, and 109%, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.     

Mouse cloning with nucleus donor cells of different age and type

We have tested different cell types as sources for nucleus donors to determine differences in cloning efficiency. When donor nuclei were isolated from cumulus cells and injected into recipient oocytes from adult hybrid mice (B6D2F1 and B6C3F1), the success rate of cloning was 1.5-1.9%. When cumulus cell donor nuclei were isolated from adult inbred mice (C57BL/6, C3H/He, DBA/2, 129/SvJ, and 129/SvEvTac), reconstructed oocytes did not develop to full term or resulted in a very low success rate (0-0.3%) with the exception of 129 strains which yielded 0.7-1.4% live young. When fetal (13.5-15.5 dpc), ovarian, and testicular cells were used as nucleus donors, 2.2 and 1.0% of reconstructed oocytes developed into live offspring, respectively. When various types of adult somatic cells (fibroblasts, thymocytes, spleen cells, and macrophages) were used, oocytes receiving thymocyte nuclei never developed beyond implantation, whereas those receiving the nuclei of other cell types did. These results indicate that adult somatic cells are not necessarily inferior to younger cells (fetal and ES cells) in the context of mouse cloning. Although fetal cells are believed to have less genetic damage than adult somatic cells, the success rate of cloning using any cell types were very low. This may largely be due to technical problems and/or problems of genomic reprogramming by oocytes rather than the accumulation of mutational damage in adult somatic cells.     

Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer

In general, oocytes arrested at metaphase of the second meiotic division (MII) are used as recipient cytoplasts for nuclear transfer (NT) procedures. MII oocytes contain high levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), which cause nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and have been implicated in nuclear reprogramming. However, the occurrence of NEBD and the extent of PCC are variable between individual oocytes and species and are dependent on donor cell type and cell cycle stage. Enucleation, which removes oocyte cytoplasm, may reduce MPF and MAPK activities and reduce reprogramming; conversely, increasing kinase activities may increase reprogramming. We compared the effects of enucleation of ovine oocytes at anaphase/telophase of the first meiotic division (AI-TI) and at MII. MPF and MAPK activities were maximal at MII; blind enucleation at AI-TI was more efficient than at MII and removed a smaller volume of cytoplasm. Neither protocol significantly affected the activity of either kinase and the fate of the donor nucleus; however, enucleation per se significantly reduced the occurrence of NEBD in NT embryos. Treatment with 10 mM caffeine significantly increased the activities of both kinases and the occurrence of NEBD but did not affect the frequency of development to the blastocyst stage; however, a significant increase in total cell numbers was observed. The results show that caffeine can increase MPF and MAPK activities in ovine oocytes and that this may contribute to an increased reprogramming in NT embryos.     

体细胞来源及培养代数对核移植重构胚发育的影响

为探讨体细胞来源及培养代数对核移植重构胚发育的影响,实验采用电融合法将小鼠2—细胞胚胎卵裂球、胚胎干细胞(ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示：2—细胞胚胎卵裂球供核重构胚发育最好,囊胚率为7．4％;ES细胞重构胚虽然发育率低,但仍有囊胚出现,比例为0．7％;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚,比例为0．2％;精原细胞重构胚只能发育到8-细胞阶段,比例为0．3％;其他几类细胞重构胚则仅能发育至4-细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第3代外都可发育到8-细胞阶段,且发育率差异不显著,但第一代细胞重构胚2-细胞发育率(40．7％)显著低于2、3和4代细胞重构胚。结果表明：不同分化程度的细胞核移植后,重新编程的难易程度是不一样的,分化程度越高则重新编程越难;未调整细胞周期的ES细胞由于多数处于S期,所以重构胚发育率很低;体外培养传代有利于体细胞核移植后重新编程。     

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.