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。     

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.     

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).     

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。     

The use ofXenopus oocytes and embryos as a route towards cell replacement

When nuclei of somatic cells are transplanted to enucleated eggs ofXenopus, a complete reprogramming of nuclear function can take place. To identify mechanisms of nuclear reprogramming, somatic nuclei can be transplanted to growing meiotic oocytes ofXenopus, and stem cell genes activated without DNA replication. The combination of somatic cell nuclear transfer with morphogen signalling and the community effect may lead towards the possibility of cell replacement therapy. When mechanisms of nuclear reprogramming are understood, it may eventually be possible to directly reprogramme human somatic cell nuclei without the use of eggs.     

Reprogramming and epigenesis

The fact that the nucleus of a differentiated somatic cell can be reprogrammed in order to sustain embryonic development is now well established. Experiments of somatic cell nuclear transfer (cloning) have proved that a foreign nucleus introduced into an enucleated oocyte can give rise to physiologically normal offsprings, with a normal lifespan. Such evidence of genome expression plasticity is also observed experimentally with heterokaryons, created by the fusion or the nuclear transfer between two somatic cells, where differentiated nuclei are able to express genes characteristic of the host cell. However, the epigenetic mechanisms that permit nuclear plasticity remain poorly understood. In this paper we present the main evidences showing important modifications of the large scale organisation of chromosomal domains and of the DNA methylation pattern upon nuclear transfer and during the first cleavages. These modifications of epigenetic marks, brought by an intimate contact between the chromatin and the recipient oocyte cytoplasmic factors, appear essential for further development. They are established over the first cell cycles of development. The onset of embryonic genome activation and the first cellular differentiation events that occur over the implantation period are two additional check-points of reprogramming that appear to be also highly dependent on epigenetic alterations. Beyond those stages, defective placental functions might be directly responsible for the fetal and postnatal physiopathologies frequently observed in cloned animals. No direct link between preimplantation reprogramming defaults, placental dysfunctions and low development to term has been established yet. The epigenetics studies which are now used to characterise loci specific and probably genotype dependent alterations in cloned animals of different species will provide invaluable help to define the role of epigenesis in the achievement of a developmental program.     

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.     

Bovine oocyte cytoplasm supports nuclear remodeling but not reprogramming of murine fibroblast cells

Nuclear transfer (NT) is used to elucidate fundamental biological issues such as reversibility of cell differentiation and interactions between the cytoplasm and nucleus. To obtain an insight into interactions between the somatic cell nucleus and oocyte cytoplasm, nuclear remodeling and gene expression were compared in bovine oocytes that had received nuclei from bovine and mouse fibroblast cells. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from mouse fibroblasts did not develop beyond the 8-cell stage. Similar nuclear remodeling procedures were observed in oocytes reconstructed with mouse and bovine fibroblast cells. Foreign centrosomes during NT were introduced into embryos reconstructed with both fibroblast cell types. A number of housekeeping mouse genes (hsp70, bax, and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells.     

Toti-/pluripotential stem cells and epigenetic modifications

The recent fascinating breakthrough in the area of stem cell research is the successful production of cloned animals via nuclear transplantation of somatic nucleus by intrinsic trans-acting factors of oocytes and trans-differentiation of somatic stem cells from adult organs induced by extrinsic growth factors. During the process of nuclear reprogramming, epigenetic modification of the somatic nuclei must be achieved to acquire toti-/pluripotential competence. However, the molecular mechanism involved is largely unknown. It has been shown that DNA methylation, histone acetylation and chromatin structure are involved in the establishment of epigenetic modification. Now it is evident that they function cooperatively to establish and maintain active or inactive chromatin state. Here we discuss the mechanisms of epigenetic modification potentially involved in the event of nuclear reprogramming.     

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 transplantation in mouse embryos

The ability of foreign nuclei to support development in nuclear transplantation manipulations has proven an effective means to assess the consequences of nuclear differentiation. In addition, nuclear transplantation might serve to define the persistence and role of maternally inherited cytoplasmic constituents during embryogenesis. We have extended the use of a technique that enables the efficient transfer of one-cell-stage pronuclei into the cytoplasm of enucleated mouse embryos, and have successfully transferred two-, four-, eight-cell-stage and inner cell mass (ICM) cell nuclei. We have also used this technique as a means to determining that the stage-specific embryonic antigen, SSEA-3, is a cytoplasmic contribution of the unfertilized ovum. The potential value of this technique in determining the developmental capacity of nuclei from various embryonic states, and in determining nuclear/cytoplasmic origins or early embryonic gene products, is discussed.