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.     

DNA甲基化与组蛋白修饰对克隆胚发育的影响

在正常的受精、发育过程中,基因组DNA不对称的去甲基化、重新甲基化以及组蛋白修饰作用发生在整个受精和胚胎发育过程中。本文将从DNA甲基化、DNA不对称的去甲基化和组蛋白修饰作用就其原理,相互之间的关系及其对胚胎发育情况的影响作以综述,并对近年来,DNA甲基化与组蛋白修饰作用在胚胎发育过程中的研究作以总结。     

成熟促进因子对克隆重构胚核重编程的调控

成熟促进因子(maturation promoting factor,MPF)由催化亚单位P34cdc2和调节亚单位cyclin组成,对细胞周期的调控起着重要作用。目前,在核移植研究中发现：供体核在MPF的作用下发生核膜破裂(nuclear envelop breakdown．NEBD)和早熟染色体凝集(premature chromosome condensation,PCC),促进了核、质蛋白质因子的交换,有利于核重编程的进行。PCC还会对供体核的倍性及形态产生影响。     

哺乳动物核质重组胚后成性重序的机制

研究表明,供体核不完全或者不正确的后成性重序可能是核移植失败的主要原因。本篇文章综述了哺乳动物核质重组胚中存在的几种不同的后成性重序机制,包括DNA甲基化、染色质重构、基因组印记与X染色体失活、端粒维持以及其他后成性遗传机制。理解重组胚后成性重序的机制将有助于我们解决核移植技术中存在的问题,进而促进它的应用。     

延迟激活对猪克隆胚胎体外、体内发育效率的影响

为了提高猪克隆效率,获得更多的克隆猪,研究了延迟激活对猪体细胞克隆胚胎体外、体内发育的影响。研究发现,和同步融合激活方法相比,延迟激活虽然会降低克隆重构胚的融合率(P> 0. 05),但能够显著提高克隆胚胎的卵裂率(P <0. 01)和囊胚率(P <0. 05);延迟激活方法重构胚使用CB辅助激活4h,其囊胚率均极显著高于不使用CB组(P <0. 01);将克隆胚胎移植到126头受体母猪后,延迟激活组受体母猪分娩率显著高于同步激活组(P <0. 05),虽然在窝均总仔、窝均活仔、克隆效率方面没有显著差异,但延迟激活组显然获得了更多的克隆仔猪。以上结果说明,延迟激活方法能够提高猪克隆胚胎的体外、体内发育效率。     

初次卵裂时间是猪克隆胚胎发育潜能的重要标识

初次卵裂时间与哺乳动物胚胎发育潜能有关．比较了不同初次卵裂时间(20～24 h,早期;25～36 h,中期;37～48 h,晚期;20～48 h,对照)的猪孤雌(parthenogenetic,PA)、体细胞核移植(somatic cell nuclear transfer,SCNT)胚胎的囊胚发育率、扩张囊胚发育率和囊胚细胞数,评价其体外发育能力．发现早期卵裂的PA胚胎发育到第6天的囊胚发育率显著高于中期、晚期以及对照组(P < 0.05;54.0% vs. 19.6%,5.4%,18.7%)．扩张囊胚发育率,早裂胚胎同样优于其他组．早期卵裂的SCNT胚胎发育到第6天的囊胚比率高于中期卵裂胚胎(32.2% vs. 23.5%),而晚期卵裂胚胎发育到囊胚的比率最低(6.3%)．早期卵裂的SCNT胚胎发育到第6天的扩张囊胚比率显著高于其余各组 (P < 0.05;18.9% vs. 5.9%、3.1%、7.4%)．囊胚细胞数在早期、中期、晚期三组之间表现出下降趋势．将早期卵裂的SCNT胚胎与未经挑选的对照组胚胎分别进行移植,观察其体内发育能力．移植早裂SCNT胚胎的受体在产仔数和克隆效率上均明显高于未经挑选胚胎的受体(4.7 vs. 2.1;3.9% vs. 0.9%),说明早裂胚胎着床后具有更强的发育能力．以上结果表明：初次卵裂时间可以作为猪克隆胚胎发育潜能的重要标识,选择早裂的胚胎进行移植,有助于提高克隆效率．     

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.     

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.     

Trichostatin A Improved Epigenetic Modifications of Transfected Cells but did not Improve Subsequent Cloned Embryo Development

Reprogramming impairment of DNA methylation may be partly responsible for the low efficiency in somatic cell nuclear transfer. In this study, bovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was dose dependent. When the TSA concentration was over 5 ng/ml, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P < 0.01). The number of cells in the S phase was significantly decreased in the 5- to 50-ng/ml TSA-treated groups, while the majority of the cells were at the G0/G1 phases. The number of eGFP-expressed cells were approximately twofold higher in 25-ng/ml (30.5%) and 50-ng/ml (29.5%) TSA groups than the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50 ng/ml of TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50 ng/ml of TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation, and eGFP gene expression was activated. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development; however, transgene expression was improved in cloned embryos.     

Gene-Specific DNA Methylation Association with Serum Levels of C-Reactive Protein in African Americans

A more thorough understanding of the differences in DNA methylation (DNAm) profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP), an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.     

The Aggregation of Four Reconstructed Zygotes is the Limit to Improve the Developmental Competence of Cloned Equine Embryos

Embryo aggregation has been demonstrated to improve cloning efficiency in mammals. However, since no more than three embryos have been used for aggregation, the effect of using a larger number of cloned zygotes is unknown. Therefore, the goal of the present study was to determine whether increased numbers of cloned aggregated zygotes results in improved in vitro and in vivo embryo development in the equine. Zona-free reconstructed embryos (ZFRE''s) were cultured in the well of the well system in four different experimental groups: I. 1x, only one ZFRE per microwell; II. 3x, three per microwell; III. 4x, four per microwell; and IV. 5x, five ZFRE''s per microwell. Embryo size was measured on day 7, after which blastocysts from each experimental group were either a) maintained in culture from day 8 until day 16 to follow their growth rates, b) fixed to measure DNA fragmentation using the TUNEL assay, or c) transferred to synchronized mares. A higher blastocyst rate was observed on day 7 in the 4x group than in the 5x group. Non-aggregated embryos were smaller on day 8 compared to those aggregated, but from then on the in vitro growth was not different among experimental groups. Apoptotic cells averaged 10% of total cells of day 8 blastocysts, independently of embryo aggregation. Only pregnancies resulting from the aggregation of up to four embryos per microwell went beyond the fifth month of gestation, and two of these pregnancies, derived from experimental groups 3x and 4x, resulted in live cloned foals. In summary, we showed that the in vitro and in vivo development of cloned zona-free embryos improved until the aggregation of four zygotes and declined when five reconstructed zygotes were aggregated.     

植物DNA甲基化及其表观遗传作用

表观遗传学(epigenetics)是研究没有DNA序列变化的、可遗传的基因表达的改变。目前研究表明,表观遗传学在植物生长发育过程中起着极其重要的作用,主要通过包括DNA甲基化、RNA干涉、基因组印记、转基因沉默等多个方面来调控植物的生长发育。其中,DNA甲基化是表观遗传学的最重要研究内容之一,是调节基因组功能的重要手段。现对植物DNA甲基化的特征、维持机制、调控机制、表观遗传作用及其研究方法进行简要论述。     

延迟激活对猪克隆胚胎体外、体内发育效率的影响

为了提高猪克隆效率,获得更多的克隆猪,研究了延迟激活对猪体细胞克隆胚胎体外、体内发育的影响。研究发现,和同步融合激活方法相比,延迟激活虽然会降低克隆重构胚的融合率(P 0. 05),但能够显著提高克隆胚胎的卵裂率(P 0. 01)和囊胚率(P 0. 05);延迟激活方法重构胚使用CB辅助激活4h,其囊胚率均极显著高于不使用CB组(P 0. 01);将克隆胚胎移植到126头受体母猪后,延迟激活组受体母猪分娩率显著高于同步激活组(P 0. 05),虽然在窝均总仔、窝均活仔、克隆效率方面没有显著差异,但延迟激活组显然获得了更多的克隆仔猪。以上结果说明,延迟激活方法能够提高猪克隆胚胎的体外、体内发育效率。     

Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage

The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P?相似文献   

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.