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．     

抗肿瘤药物对小鼠早期胚胎发育的影响

表观遗传修饰在基因表达和克隆胚胎的早期发育方面有重要作用.表现遗传修饰至少发生在两个关键时期--配子形成期和植入前胚胎,如果在此期间发生异常,则会导致胚胎的死亡及出生后各种疾病的发生.其中DNA的甲基化是最重要的一种表观遗传修饰类型,DNA甲基化在哺乳动物发育过程中起关键作用.综述几种类型抗肿瘤药物作用机制——其使胚胎的DNA甲基化降低,引起转录活性降低,进而导致胚胎发育停滞.     

转基因克隆牛胎盘中印迹基因PEG10的DNA甲基化水平

低效率的体细胞核移植技术显著制约着该技术在转基因动物生产上的广泛应用。目前认为供体细胞核不能被受体卵母细胞胞质完全的表观重编程是其效率低下的最主要原因,而DNA甲基化是基因表观修饰的主要方式之一。为了探求转基因克隆牛的死亡是否与其胎盘中印迹基因的甲基化的重编程程度相关,文章通过亚硫酸氢盐测序法(Bisulfite sequencing PCR,BSP)和亚硫酸氢盐联合限制性内切酶分析法(Combined bisulfite restriction analysis,COBRA),对印迹基因PEG10在围产期死亡且存在发育缺陷的转基因克隆牛的胎盘(死亡组)和存活的转基因克隆牛的胎盘(存活组)与正常对照牛胎盘(对照组)的DNA甲基化水平进行了详细的比较。结果发现,与对照组相比,PEG10基因在死亡组上表现出异常的超甲基化水平,而存活组与对照组相比无显著性差异。研究结果显示,胎盘中印迹基因的DNA甲基化表观重编程不彻底可能是导致转基因克隆牛发育异常进而死亡的主要原因之一。     

山羊克隆胎儿不同组织中H19基因CpG岛甲基化模式及表达量检测

表观重编程异常是核移植胚胎发育异常的重要原因。为了研究克隆山羊胎儿不同组织中H19基因CpG岛甲基化水平和相对表达量,本实验运用亚硫酸盐法和荧光实时定量PCR法分别检测了死亡克隆山羊胎儿和同期普通山羊胎儿(对照组)肝脏、胎盘、肾脏、肺脏和心脏组织中H19基因CpG岛甲基化水平和mRNA的相对表达量。结果表明,克隆山羊胎儿胎盘组织中H19基因第5个CpG岛的甲基化水平显著高于对照组(70%vs49.41%,P0.05),H19基因相对表达量显著低于对照组(883.3vs1264.5,P0.05);肺脏组织甲基化水平显著低于对照组(63.53%vs88.24%,P0.05),相对表达量显著高于对照组(1003.4vs515.5,P0.05);其他各组差异不显著(P0.05)。结果说明,H19基因在克隆山羊胎儿部分组织中DNA甲基化重编程异常,而且这种异常影响H19基因的正常表达,这也可能是导致克隆动物死亡的重要因素之一。     

体细胞核移植胚胎核重编程的研究进展

尽管在多种哺乳动物种系中成功制备了体细胞克隆后代,但当前的克隆技术仍有许多亟待解决的问题。体细胞核移植胚胎大多存在许多发育异常,造成了妊娠早期高流产率和出生后高死亡率。有研究认为,克隆胚胎发育障碍的一个重要的原因是供体细胞的遗传重编程不完全。哺乳动物种系中,DNA甲基化是胚胎发育期转录调节的必需步骤,除了单拷贝基因序列外,在基因组很多的区域都可以观测到克隆胚胎的异常甲基化。此外,克隆胚胎的基因印迹也存在异常。     

胚胎发育过程中的DNA甲基化作用

哺乳动物的正常发育取决于表观遗传学调控机制准确无误地运行.其中尤为重要的是发生在原生殖细胞和胚胎中的基因组范围内的DNA甲基化模式重排等表观遗传学修饰.胚胎发育过程中的DNA甲基化作用与基因印记的建立、基因表达的调控以及细胞和胚胎的形态建成都密切相关.DNA甲基化发生机制和功能的阐明将对哺乳动物个体发育与人类疾病研究有重要意义.     

综述: DNA甲基化在动物胚胎和生殖细胞发育过程中的重编程

表观遗传信息DNA甲基化在动物的发育、细胞分化和器官形成过程中,起着至关重要的作用．近期,关于DNA甲基化在脊椎动物胚胎发育和生殖细胞发育过程重编程的研究取得了重要的进展．发现斑马鱼的早期胚胎完整地继承了精子的DNA甲基化图谱,而哺乳动物的早期胚胎和原始生殖细胞发育过程则经历了整体去甲基化并重新建立甲基化图谱的过程,但胚胎发育过程中基因的印迹区未发生DNA去甲基化,而生殖细胞发育过程中印迹区的甲基化修饰被消除．     

DNA甲基化在动物胚胎和生殖细胞发育过程中的重编程

表观遗传信息DNA甲基化在动物的发育、细胞分化和器官形成过程中,起着至关重要的作用.近期,关于DNA甲基化在脊椎动物胚胎发育和生殖细胞发育过程重编程的研究取得了重要的进展.发现斑马鱼的早期胚胎完整地继承了精子的DNA甲基化图谱,而哺乳动物的早期胚胎和原始生殖细胞发育过程则经历了整体去甲基化并重新建立甲基化图谱的过程,但胚胎发育过程中基因的印迹区未发生DNA去甲基化,而生殖细胞发育过程中印迹区的甲基化修饰被消除.     

表观遗传调控在哺乳动物体细胞核移植上的应用研究进展

体细胞核移植也称为克隆,是指将体细胞经显微操作植入到去核的卵母细胞质中,不通过精子和卵子结合,在体外生产出与供体细胞具有相同遗传物质的胚胎以及动物个体。近年来克隆技术不断发展,但其效率依然有待提高,导致克隆效率低下的重要原因之一便是供体细胞核物质表观遗传重编程不完全。DNA甲基化和组蛋白乙酰化是人们研究最多的两种表观遗传修饰,此外,基因印记、X染色体失活和端粒长度异常修饰状态也受到研究者的关注。因此如何调节和修复植入前克隆胚胎异常的表观遗传学状态对于提高克隆效率有着重要的意义。本综述植入前克隆胚胎存在的异常表观遗传现象,为建立更为高效的体细胞核移植体系提供一定的理论依据。     

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。     

DNA methylation variation in cloned mice

Summary: Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue‐specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue‐specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue‐specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned. genesis 30:45–50, 2001. © 2001 Wiley‐Liss, Inc.     

Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1-04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1-03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.     

Aberrant DNA methylation imprints in aborted bovine clones

Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.     

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.