Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.     

Cloning of endangered mammalian species: any progress?

Attempts through somatic cell nuclear transfer to expand wild populations that have shrunk to critical numbers is a logical extension of the successful cloning of mammals. However, although the first mammal was cloned 10 years ago, nuclear reprogramming remains phenomenological, with abnormal gene expression and epigenetic deregulation being associated with the cloning process. In addition, although cloning of wild animals using host oocytes from different species has been successful, little is known about the implication of partial or total mitochondrial DNA heteroplasmy in cloned embryos, fetuses and offspring. Finally, there is a need for suitable foster mothers for inter-intra specific cloned embryos. Considering these issues, the limited success achieved in cloning endangered animals is not surprising. However, optimism comes from the rapid gain in the understanding of the molecular clues underlying nuclear reprogramming. If it is possible to achieve a controlled reversal of the differentiated state of a cell then it is probable that other issues that impair the cloning of endangered animals, such as the inter-intra species oocyte or womb donor, will be overcome in the medium term.     

核移植后的细胞核再程序化机制研究进展

目前动物克隆技术体系极待完善,其极低的成功率及克隆动物普遍存在的早衰、早天现象是阻碍研究深入进行的首要问题,其突破的关键在于对核移植后的细胞核再程序化机制的阐明。从移植核在结构上的重塑、移植核与受体卵细胞质所处的细胞周期及其相互作用、重构胚与两性胚在分子水平的变化等多方面研究表明：受体细胞质的环境对于细胞核的再程序化至关重要,处于有丝分裂各时期的细胞作为核供体一旦移植到卵母细胞后,移植核在卵质环境里将出现结构上的重塑和分子的再程序化;移植核与受体卵问细胞周期的相容性、重构胚的染色体倍性的正确与否,可能是决定重构胚发育率高低的重要因素;合子型基因激活是基因表达再程序化的关键事件之一;印记基因对于体细胞克隆动物移植核的再程序化过程可能起着非常独特的作用。     

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.     

Production of a cloned calf using zona-free serial nuclear transfer

The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.     

克隆动物发育过程中基因组的重编程

自克隆羊“多莉”诞生后利用体细胞核移植技术进行克隆动物的研究已取得很大进展,体细胞克隆的牛、猪、山羊、猫、兔等已陆续出生,但克隆动物的成活率一直都比较低,并且产出的动物大部分存在某种程度的缺陷.最新研究表明,克隆动物胚胎基因组的重编程出现偏差和失误,尤其是去甲基化不足可能是核克隆动物出现异常的关键所在.探讨早期克隆胚胎重编程,特别是对DNA的甲基化,以及供体核在受体卵胞质中进行核重组,为研究克隆胚胎发育和解决克隆动物中的两大难题——即基因组的重编程和核质相互作用提供一些线索.     

Nuclear transfer: Progress and quandaries

Cloning mammals by nuclear transfer is a powerful technique that is quickly advancing the development of genetically defined animal models. However, the overall efficiency of nuclear transfer is still very low and several hurdles remain before the power of this technique will be fully harnessed. Among these hurdles include an incomplete understanding of biologic processes that control epigenetic reprogramming of the donor genome following nuclear transfer. Incomplete epigenetic reprogramming is considered the major cause of the developmental failure of cloned embryos and is frequently associated with the disregulation of specific genes. At present, little is known about the developmental mechanism of reconstructed embryos. Therefore, screening strategies to design nuclear transfer protocols that will mimic the epigenetic remodeling occurring in normal embryos and identifying molecular parameters that can assess the developmental potential of pre-implantation embryos are becoming increasingly important. A crucial need at present is to understand the molecular events required for efficient reprogramming of donor genomes after nuclear transfer. This knowledge will help to identify the molecular basis of developmental defects seen in cloned embryos and provide methods for circumventing such problems associated with cloning the future application of this technology.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming?

Fertile offspring from somatic cell nuclear transfer (SCNT) is the goal of most cloning laboratories. For this process to be successful, a number of events must occur correctly. First the donor nucleus must be in a state that is amenable to remodeling and subsequent genomic reprogramming. The nucleus must be introduced into an oocyte cytoplasm that is capable of facilitating the nuclear remodeling. The oocyte must then be adequately stimulated to initiate development. Finally the resulting embryo must be cultured in an environment that is compatible with the development of that particular embryo. Much has been learned about the incredible changes that occur to a nucleus after it is placed in the cytoplasm of an oocyte. While we think that we are gaining an understanding of the reorganization that occurs to proteins in the donor nucleus, the process of cloning is still very inefficient. Below we will introduce the procedures for SCNT, discuss nuclear remodeling and reprogramming, and review techniques that may improve reprogramming. Finally we will briefly touch on other aspects of SCNT that may improve the development of cloned embryos.     

鱼类核移植与重编程

鱼类核移植是动物克隆研究的一个重要领域, 我国学者在上世纪60年代首创了鱼类的核移植研究。以斑马鱼为模式动物, 进行核移植与再程序化研究具有独特的优势。文章总结了鱼类细胞核移植研究的历史、斑马鱼核移植研究概况、以及影响核移植胚胎发育的因素, 特别是核移植胚胎基因组的表观遗传修饰, 如基因组DNA甲基化及组蛋白乙酰化和甲基化等的研究, 将有助于完善克隆技术并提高克隆的成功率, 推动克隆技术的广泛开展和应用。     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

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。     

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44–47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT.     

Asymmetric nuclear reprogramming in somatic cell nuclear transfer?

Despite the progress achieved over the last decade after the birth of the first cloned mammal, the efficiency of reproductive cloning remains invariably low. However, research aiming at the use of nuclear transfer for the production of patient-tailored stem cells for cell/tissue therapy is progressing rapidly. Yet, reproductive cloning has many potential implications for animal breeding, transgenic research and the conservation of endangered species. In this article we suggest that the changes in the epi-/genotype observed in cloned embryos arise from unbalanced nuclear reprogramming between parental chromosomes. It is probable that the oocyte reprogramming machinery, devised for resident chromosomes, cannot target the paternal alleles of somatic cells. We, therefore, suggest that a reasonable approach to balance this asymmetry in nuclear reprogramming might involve the transient expression in donor cells of chromatin remodelling proteins, which are physiologically expressed during spermatogenesis, in order to induce a male-specific chromatin organisation in the somatic cells before nuclear transfer.