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。     

后生遗传修饰及其对动物克隆的影响

近年来,不断有新的哺乳动物和两栖类动物被成功克隆,但这并不能掩盖克隆效率过低和克隆动物异常的现实,为了解决这一问题,人们对克隆机理进行了大量研究。高度分化的体细胞核在去核的卵质中去分化和再程序化不完全是导致动物克隆失败的主要原因,而去分化和再程序化不完全主要是由于基因组去甲基化不充分和过早再甲基化引起克隆胚中甲基化水平比正常胚中偏高所至,这可引起一些重要基因的异常表达,尤其是印记基因。这些机制的研究对提高克隆效率有着重要意义。     

小鼠卵细胞质对供体核DNA甲基化和U2afbp-rs基因表达的调控

利用细胞核移植技术将NIH3T3细胞核和孤雌桑椹胚单个卵裂球,分别移植到去核MⅡ期受体卵母细胞中,通过免疫荧光染色后比较体外受精胚、孤雌胚、NIH3T3核移植重组胚和孤雌桑椹胚核移植重组胚附植前各时期胚胎DNA甲基化水平的变化,以探明克隆胚细胞核去分化与DNA甲基化的相互关系.利用Real-timePCR技术检测体外受精胚、孤雌胚和孤雌桑椹胚核移植重组胚附植前各时期胚胎中,印记基因U2afbp-rs基因以及非印记基因eIF-4C基因表达量的变化,以探明小鼠卵细胞质对克隆胚细胞核中印记基因表达的调控.结果表明,克隆胚供体核基因组DNA在核移植后并没有发生主动去甲基化.孤雌桑椹胚核移植后重组胚中U2afbp-rs基因和eIF-4C基因的表达水平要显著低于对照孤雌胚,但其表达量变化规律与对照孤雌胚相同,说明了卵细胞质对供体核印记基因的表达具有一定的调控作用.     

Mash2基因在体细胞克隆牛肺脏中DNA甲基化状态分析

体细胞核移植(体细胞克隆)技术在动物生产、医药工业、治疗性克隆以及对珍稀濒危动物的拯救有重要意义,然而克隆效率低下以及克隆动物发育异常,严重制约了克隆技术的发展和应用．在体细胞核克隆中,供体核来自高度分化了的体细胞,发生在核移植后几小时内供体核的重编程,决定了克隆胚胎的发育能力．印记基因是由等位基因表观遗传修饰的不对称导致的基因表达具有亲本选择性,而DNA甲基化是调控印记的一个主要方式．印记基因Mash2在胚胎发育和器官形成过程中起着非常重要的作用．为了探求核移植过程中Mash2基因DNA 甲基化的表观重编程是否充分,利用亚硫酸氢盐测序法对出生48 h内死亡的体细胞核移植牛和正常对照牛肺脏中Mash2基因的DNA甲基化状态进行分析．结果显示,尽管位于Mash2基因启动子和第一个外显子处的CpG岛在正常牛和克隆牛中甲基化水平都不高(20.04%,5.55%),但克隆组的甲基化水平仍显著低于正常对照组 (P < 0.05)．甲基化模式正常组中9N3有5种不同的形式,9N4仅1种;而克隆组9C3和9C5也分别是1种．推测Mash2基因的异常DNA甲基化很可能是导致克隆牛肺脏发育异常的一个重要原因．     

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。     

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.     

外源基因在鱼类核移植胚胎中的转录起始

比较了hGH转植基因在F4代的转MThGH基因鱼, F4代胚胎细胞的核移植后代, 以及F4代尾鳍培养细胞的核移植后代中的转录时序差异. RT-PCR实验结果表明, hGH基因在转基因鱼F4代胚胎中从原肠早期开始转录; F4代胚胎细胞核移植的后代在囊胚早期已能检测到hGH基因转录本; F4代尾鳍培养细胞核移植的后代自16胞期就出现hGH基因的转录.上述结果表明, 鲤鱼卵细胞质对分化细胞核特定基因的再程序化能力是有限的, 进而推论鱼类细胞核移植试验中仅有少部分的供体核能够发生完全再程序化.     

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。     

体细胞核移植的研究进展

自从克隆羊多利问世以后,体细胞核移植有了较大的发展,陆续有新的动物克隆成功,但克隆的效率仍然较低。本文综述了体细胞核移植技术研究的进展情况,介绍了基因背景、核移植步骤、胚胎相关支持技术对核移植成功率的影响以及核移植技术中的基因修饰和对核重新程序化的最新认识。     

哺乳动物基因组印记的擦除、建立和维持机理

基因组印记主要依靠印记基因DNA甲基化方式调控,这种表观遗传修饰让多种哺乳动物出现基因单等位表达现象。印记的擦除发生在原始生殖细胞(primordial germ cells,PGCs)时期,其主要途径为活化诱导的胞苷脱氨酶(activation-induced cytidine deaminase,AID)、TET(ten-eleven translocation)蛋白介导的去甲基化。印记的建立发生在配子发生期,雌雄有明显的不同。印记的维持在多种因子的共同作用下完成,主要参与的蛋白有Dnmt1、Dppa3、KAP1和ZFP57等。印记的维持贯穿整个发育阶段,并通过细胞分裂遗传给子代。机体正常生长发育有赖于印记基因的正常表达。随着第一个印记基因IGF2R的发现,对于印记机制的研究不断推进。该文将概述基因组印记的建立、维持、擦除机理以及克隆动物中存在的印记基因异常重编程。     

Nuclear fragility,blaming the blebs

Although textbook pictures depict the cell nucleus as a simple ovoid object, it is now clear that it adopts a large variety of shapes in tissues. When cells deform, because of cell crowding or migration through dense matrices, the nucleus is subjected to large constraints that alter its shape. In this review, we discuss recent studies related to nuclear fragility, focusing on the surprising finding that the nuclear envelope can form blebs. Contrary to the better-known plasma membrane blebs, nuclear blebs are unstable and almost systematically lead to nuclear envelope opening and uncontrolled nucleocytoplasmic mixing. They expand, burst, and repair repeatedly when the nucleus is strongly deformed. Although blebs are a major source of nuclear instability, they are poorly understood so far, which calls for more in-depth studies of these structures.     

Composition,structure and function of HeLa cell nuclear envelope

Summary Nuclear envelope pieces were isolated from HeLa cells, and their structure was investigated by using the negative staining technique. Structural data such as for pore diameters, pore frequency and the frequency of central granules within the pores are presented. In addition, substructural details of pore complexes as revealed by the technique employed are described. The results obtained from HeLa are compared with those of nuclear envelopes similarly prepared from diverse other kinds of cells including non-tumorous cells from human source (fetal lung fibroblasts).The authors thank Drs. H. Falk, H. Kleinig, U. Scheer, and F. Wunderlich for helpful discussions and Miss Marianne Winter for skilful technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft.     

Nuclear structure and intranuclear retention of premature RNAs

Eukaryotic cells are highly compartmentalized, each compartment being surrounded by a lipid bilayer. This membrane-based organization allows cells to use their volumes to encode information. The lack of intranuclear membranes suggested that the nucleus was largely devoid of structural organization. However, recent work has defined numerous specialized nuclear subdomains. Importantly, RNA processing factors do not display random distribution but cluster in defined nuclear bodies. Although these structures are well characterized morphologically, their function in relation to RNA metabolism remains elusive. In this review, we will discuss the putative participation of nuclear substructures in a quality control step of RNA biogenesis, the nuclear retention of premature RNA.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.     

On the universality of nuclear pore complex structure

Summary Substructural details of the nuclear pore complex were studied in diverse plant and animal cells with both section technique and negative staining of isolated nuclear envelope pieces. The structures observed after the different techniques, including a variety of fixation procedures, are compared and their significance is discussed. It is shown that, down to the 15–20 Å level, the architecture of the nuclear pore complex is universal among such diverse cell types as from, e. g., onion root tips, bean leaves, mammalian liver parenchyma, HeLa cell cultures, and amphibian germ material. The fundamental substructures of the pore complex such as (1) the annular granules, (2) the fibrils attached to the annuli, (3) the central granules, (4) the fibrils in the pore interior including those which make up the inner ring and/or those which connect the central granule to the pore margin, are recognized in all cell types studied. The dynamic variability of the central granule morphology is emphasized and observations are presented which suggest that the view of such centrally located material as representing ribonucleoproteins in a transitory state of nucleocytoplasmic migration can be extended to generality. General concepts of the nuclear pore complex structure are presented as alternative model views revealing either a more compact, predominantly granular, or a more fibrillar aspect.The author gratefully acknowledges the frequent discussions and cooperation with his team-colleagues Drs. H. Falk (in the work on leaf material) and U. Scheer (in working with amphibian oocytes) as well as the skillful technical assistance of Miss Marianne Winter and Miss Sigrid Krien. The work was supported by the Deutsche Forschungsgemeinschaft.     

A Crowdsourced nucleus: Understanding nuclear organization in terms of dynamically networked protein function

The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear function. This compartmentalization involves genome organization as well as the formation of nuclear bodies and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a common theme for nuclear proteins is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information technology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear organization and function. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.     

NSort/DB:An Intranuclear Compartment Protein Database

Distinct substructures within the nucleus are associated with a wide variety of important nuclear processes.Structures such as chromatin and nuclear pores have specific roles,while others such as Cajal bodies are more functionally varied.Understanding the roles of these membraneless intra-nuclear compartments requires extensive data sets covering nuclear and compartment-associated proteins.NSort/DB is a database providing access to intra-or sub-nuclear compartment associations for the mouse nuclear proteome.Based on resources ranging from large-scale curated data sets to detailed experiments,this data set provides a high-quality set of annotations of non-exclusive association of nuclear proteins with structures such as promyelocytic leukaemia bodies and chromatin.The database is searchable by protein identifier or compartment,and has a documented web service API.The search interface,web service and data download are all freely available online at http://www.nsort.org/db/.Availability of this data set will enable systematic analyses of the protein complements of nuclear compartments,improving our understanding of the diverse functional repertoire of these structures.