Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.     

核移植与供体细胞

“多莉”羊的诞生是生物界的一个里程碑,它之所以引起如此大的轰动主要是因为它来源于培养的成年绵羊乳腺上皮细胞,这是人类第一次证明分化的体细胞可以被重编程后恢复全能性并最终分化发育成一个动物个体。这说明哺乳动物分化的体细胞核仍具有全套的遗传物质并能够被卵母细胞逆转恢复全能性。然而,关于多莉的供体细胞来源却一直是克隆领域的一个谜。由于体细胞克隆的效率非常低,而用于核移植的供体细胞悬液中往往含有多种类型的细胞,这使得我们很难确切地知道最终获得的克隆动物是来源于哪一种细胞。这种不确定性给我们研究核移植诱导体细胞重编程的机制带来了很大的困难,因此,对供体细胞的研究也是核移植研究领域的一个重要课题,这包括各种组织来源的体细胞是否均可以用于核移植,终末分化的体细胞是否能够用于核移植,组织干细胞是否更有利于体细胞重编程,供体细胞的分化状态是否与核移植的效率有关,死亡的体细胞是否也可以用于核移植等等。本文综述了核移植中与供体细胞相关的最新研究进展。     

核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。     

SMC complexes and topoisomerase II work together so that sister chromatids can work apart

The pairing of sister chromatids in interphase facilitates error-free homologous recombination (HR). Sister chromatids are held together by cohesin, one of three Structural Maintenance of Chromosomes (SMC) complexes. In mitosis, chromosome condensation is controlled by another SMC complex, condensin, and the type II topoisomerase (Top2). In prophase, cohesin is stripped from chromosome arms, but remains at centromeres until anaphase, whereupon it is removed via proteolytic cleavage. The third SMC complex, Smc5/6, is generally described as a regulator of HR-mediated DNA repair. However, cohesin and condensin are also required for DNA repair, and HR genes are not essential for cell viability, but the SMC complexes are. Smc5/6 null mutants die in mitosis, and in fission yeast, Smc5/6 hypomorphs show lethal mitoses following genotoxic stress, or when combined with a Top2 mutant, top2-191. We found these mitotic defects are due to retention of cohesin on chromosome arms. We also show that Top2 functions in the cohesin cycle, and accumulating data suggests this is not related to its decatenation activity. Thus the SMC complexes and Top2 functionally interact, and any DNA repair function ascribed to Smc5/6 is likely a reflection of a more fundamental role in the regulation of chromosome structure.     

Nuclear transfer in practice

The technique of nuclear transfer (NT) allows the production of embryos, fetuses, and offspring from a range of embryonic, fetal, and adult derived cell types in a range of species. Successful development is dependent upon numerous factors, including type of recipient cell, source of recipient cell, method of reconstruction, activation, embryo culture, donor cell type, and donor and recipient cell cycle stages. The present review will discuss the uses of NT, the techniques presently available, and the factors affecting subsequent development.     

T cells work together to fight cancer.

Recent studies have identified new melanoma antigens that are recognised by CD4(+) T cells. Analysis of tumour-specific CD4(+) T-cell responses may lead to the development of optimal anti-cancer vaccines that can induce an orchestrated effort of tumour-specific CD4(+) and CD8(+) T cells in the fight against cancer.     

Mineralocorticoids and cerebral angiotensin may act together to produce sodium appetite

The sodium appetite that follows sodium deficiency may be aroused by a synergy of the hormones of sodium deficiency (angiotensin and mineralocorticoid) rather than by the deficiency itself. Recent evidence supporting this idea is discussed with emphasis on the possibility that angiotensin of cerebral origin may be more effective in this synergy than that of renal origin.     

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.     

Nuclear transfer in cats and its application

Nuclear transfer (NT) technology is typically used for generating identical individuals, but it is also a powerful resource for understanding the cellular and molecular aspects of nuclear reprogramming. Most recently, the procedure has been used in humans for producing patient-specific embryonic stem cells. The successful application of NT in cats was demonstrated by the birth of domestic and non-domestic cloned kittens at a similar level of efficiency to that reported for other mammalian species. In cats, it has been demonstrated that either in vivo or in vitro matured oocytes can be used as donor cytoplasts. The length of in vitro oocyte maturation affects in vitro development of reconstructed embryos, and oocytes matured in vitro for shorter periods of time are the preferred source of donor cytoplasts. For NT, cat somatic cells can be synchronized into the G0/G1 phase of the cell cycle by using different methods of cell synchronization without affecting the frequency of in vitro development of cloned embryos. Also, embryo development to the blastocyst stage in vitro is not influenced by cell type, but the effect of cell type on the percentage of normal offspring produced requires evaluation. Inter-species NT has potential application for preserving endangered felids, as live offspring of male and female African wildcats (AWC, Felis silvestris lybica) have been born and pregnancies have been produced after transferring black-footed cat (Felis nigripes) cloned embryos into domestic cat (Felis silvestris catus) recipients. Also, successful in vitro embryo development to the blastocyst stage has been achieved after inter-generic NT of somatic cells of non-domestic felids into domestic cat oocytes, but no viable progeny have been obtained. Thus, while cat cytoplasm induces early nuclear remodeling of cell nuclei from a different genus, the high incidence of early embryo developmental arrest may be caused by abnormal nuclear reprogramming. Fetal resorption and abortions were frequently observed at various stages of pregnancy after transfer of AWC cloned embryos into domestic cat recipients. Abnormalities, such as abdominal organ exteriorization and respiratory failure and septicemia were the main causes of death in neonatal cloned kittens. Nonetheless, several live domestic and AWC cloned kittens have been born that are seemingly normal and healthy. It is important to continue evaluating these animals throughout their lives and to examine their capability for natural reproduction.     

肠道淋巴组织与共生细菌对肠道环境稳态的共同维持

肠道环境的稳态不仅是正常消化功能的基础,还是抵御病原体入侵的重要防线。定居于肠道内的细菌与肠道免疫系统间存在着广泛的相互影响和相互补充,在维持稳态过程中发挥协同作用。本综述结合国内外相关研究进展,简要阐述肠道正常菌群对肠道淋巴组织发育的影响和对局部乃至全身免疫反应的调节机制,进而说明菌群如何与机体共同维持肠道稳态。 更多还原