体细胞克隆中核的重编程

尽管体细胞克隆在绵羊、牛、小鼠、猪、山羊、兔、猫、大鼠和骡子等物种中都获得了成功，但却未能得到狗和猕猴的克隆个体，而且克隆效率非常低，克隆效率低使体细胞克隆技术在科研和生物技术等方面的应用受到限制．供体核移入去核的卵细胞后，必须经过表观遗传修饰的重编程，回到胚胎开始发育的全能状态。目前认为：供体核的不完全重编程是导致克隆效率低的主要原因。本文从DNA甲基化、组蛋白乙酰化、X染色体失活、端粒、印记基因以及其他发育相关基因的表达几个方面来探讨影响克隆效率的因素。     

基因组表观重编程对体细胞核移植成功率的影响

基因组表观重编程缺陷是影响体细胞核移植效率的主要因素,本文讨论了表观重编程的两大主要机制——DNA甲基化及组蛋白修饰及其对体细胞核移植重构胚胎发育的影响,并综述了几种促进核重编程的方法。     

表观遗传修饰影响哺乳动物体细胞核移植效率的研究进展

表观遗传修饰是一种不依赖于DNA序列变化的可逆、可遗传修饰，在哺乳动物胚胎发育的整个阶段均可发生，是影响哺乳动物体细胞核移植效率的主要因素之一。其中，DNA甲基化、组蛋白的动态修饰、X染色体失活、端粒与端粒酶活性变化作为常见的表观遗传修饰类型，任一修饰形式的异常都会影响基因的表达，引发体细胞重编程错误导致核移植效率降低。近年来，随着体细胞核移植技术研究的不断深入，表观遗传修饰影响体细胞核移植效率的关键作用机制日益明确。本文通过综述不同类型的表观遗传修饰影响哺乳动物体细胞核移植效率的研究进展，以期在表观遗传修饰层面为提高哺乳动物体细胞核移植效率提供新思路。     

绵羊无活性体细胞核移植后发育成羔羊

随着胚胎核移植 ,特别是成年动物体细胞核移植的成功为基础研究和应用研究提供了可能性 ,但目前的核移植技术使基因组重新编程的能力是不稳定的 ,本试验所用的技术没有强有力的科学资料依据。胞核重新编程结果表明 ,胚胎和体细胞核移入去核卵母细胞内获得多能性或全能性的过程中发生了结构上和功能上的修饰。亲代基因组通过生殖细胞系发生重新编程 ,是复杂演化进程中符合逻辑的结果 ,这种演化进程用来诱导父性和母性突变间功能上的互补。当前的体细胞克隆程序走了很大捷径 ,供体核在正常细胞系中逐步发生的变化在普通的细胞质环境下 (可能在…     

哺乳动物体细胞核移植的DNA甲基化重编程研究进展

DNA甲基化是基因组主要的表观遗传修饰方式之一.核移植重构胚在对供体细胞基因组进行甲基化重编程过程中会出现异常的甲基化模式,而异常的甲基化重编程是导致克隆胚早期死亡及克隆动物发育畸形的主要原因.论文针对体细胞克隆动物基因组DNA的甲基化模式、造成克隆胚胎甲基化异常的原因及异常甲基化对重构胚胎发育的影响等进行了综述.深入研究核移植重构胚甲基化重编程的机制,有助于完善核移植技术,提高克隆效率,使其更好地应用于基础研究和生产实践.     

动物克隆技术

动物克隆技术,即动物核移植技术,是通过显微操作,电融合等实验手段,将发育到一定阶段的核供体(胚胎细胞或体细胞)的细胞核取出,注入相应发育阶段的去核的原核胚或成熟卵母细胞中,再将重构胚的胚胎移植,到相应生理状态的动物体内,达到大量生产遗传同质动物的一种生物工程技术。1动物克隆技术的应用克隆技术对畜牧业的发展将产生重大影响。利用动物克隆技术,可缩短育种年限。据报道,自1987年牛胚胎细胞移植成功,牛克隆胚胎的重复克隆已达6个世代,并获得第三代克隆牛,由一枚胚胎反复克隆已得到近200枚克隆胚胎,母牛繁殖后代数可以呈几何基数增…     

分子育种手段之微卫星技术的应用与前景

在国内,1995年,西北农业大学窦忠英等用胚胎细胞核移植产生了6头猪。1996年,湖北农科院魏庆信等得到了胚胎细胞核移植猪。1997年赵浩斌等和2000年Li等分别获得猪胚细胞核移植后代。1998年,东北农业大学的李光鹏等把猪胚胎的细胞核通过电融合移植到去核的体内和体外成熟卵母细胞中,出生了2头克隆猪。2003年,西北农林科技大学冯秀亮等得到了人体细胞和猪卵母细胞异种核移植囊胚。一个良好的遗传标记应高度稳定,具丰富的多态性,且在全基因组的分布较均匀,从简单高效的使用要求出发,又希望尽可能少的遗传标记基因库,微卫星基本符合上述要求。因…     

哺乳动物卵母细胞成熟及早期胚胎发育中组蛋白修饰研究进展

在动物生产中,卵母细胞及早期胚胎的发育质量是影响动物繁殖性能的关键因素.哺乳动物卵母细胞成熟及早期胚胎发育过程中存在活跃的组蛋白修饰变化,其中对组蛋白甲基化和乙酰化研究最多,活跃的组蛋白修饰变化在细胞重编程过程中起到重要的调控作用.本文综述了哺乳动物卵母细胞及受精后早期胚胎的组蛋白甲基化和乙酰化动态修饰变化以及影响因素...     

体细胞克隆的小鼠胚胎在附植前后发育的研究

采用成年小鼠的卵丘细胞核、胎儿成纤维细胞作核供体细胞来进行核移植,研究小鼠卵母细胞的去核程序和影响重构胚附植前发育的激活条件;随后将重构胚与供体细胞系共培养后获得囊胚阶段的小鼠重构胚,并将其移植到受体鼠体内后获得了24%克隆胚胎。结果表明,小鼠卵母细胞质能够进行重编程来支持早期的胚胎发育。     

猪体细胞克隆和基因编辑技术研究

<正>体细胞核移植（somaticcell nucleartransfer,SCNT）是一种将已分化的细胞重编程恢复其全能性,进而生产与供体细胞基因型完全相同后代的无性繁殖技术。包括收集并成熟卵母细胞,供体细胞核处理,重构胚的体外激活,胚胎的培养和移植等过程。猪的SCNT技术的建立在农业和医学有广泛的应用。     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Establishment,Differentiation, Electroporation and Nuclear Transfer of Porcine Mesenchymal Stem Cells

The limited success of somatic cell nuclear transfer (SCNT) is largely attributed to defects in epigenetic reprogramming of the donor genome. Donor cell types with distinct potential competence may offer different epigenetic flexibility for subsequent genome reprogramming in SCNT. Stem cells possibly enable their genomes to be more readily reprogrammed than differentiated cells. To improve the efficiency of cloning, porcine mesenchymal stem cells (pMSCs) were isolated and well identified by 6‐channel flow cytometry and differentiation assays and were used as donors in SCNT. Compared with porcine embryonic fibroblasts (pEFs), our results showed that pMSCs markedly enhanced cloned embryo development in terms of cleavage and blastocyst formation (p < 0.05). To enhance the epigenetic flexibility of pMSCs, classical reprogramming factors (RFs) were transfected by electroporation, and we achieved optimization with ectopic expression of RFs in pMSCs. Our results suggest that the epigenetic status of donor cells has an improvement on genome reprogramming, and multipotent pMSCs favoured subsequent embryonic development.     

Inheritance of histone H3 methylation in reprogramming of somatic nuclei following nuclear transfer

Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.     

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre‐implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.     

Trichostatin A and nuclear reprogramming of cloned rabbit embryos

To investigate the influence of histone deacetylases on nuclear reprogramming after nuclear transfer, we treated the cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA). In the present study, global changes in acetylation of histone H3-lysine 14, histone H4-lysine 12, and histone H4-lysine 5 were studied in rabbit in vivo fertilized embryos, somatic cell nuclear transfer (SCNT) embryos, and TSA-treated SCNT embryos. From the pronuclear to the morula stage, the deacetylation-reacetylation changes in acetylation of histone H3-lysine 14 and histone H4-lysine 12 occurred in both fertilized embryos and TSA-treated cloned embryos; however, the distribution pattern in untreated cloned embryos failed to display such changes. More interesting, the signal of acetylation of histone H4-lysine 12 in cloned embryos was detected in both the inner cell mass and the trophectoderm, whereas TSA-treated cloned embryos showed the same staining pattern as fertilized embryos and the staining was limited to the inner cell mass. The histone acetylation pattern of TSA-treated SCNT embryos appeared to be more similar to that of normal embryos, indicating that TSA could improve nuclear reprogramming after nuclear transfer.     

Recent progress in bovine somatic cell nuclear transfer

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full‐term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT.     

WT1基因组蛋白乙酰化修饰对细胞核重编程的影响

利用体细胞移植技术获得克隆动物的成功是几十年来生命科学领域取得的重大突破之一,这项技术引起了社会的广泛关注。然而,由于哺乳动物克隆效率低下,且克隆后代发育异常等问题,已成为目前制约动物克隆技术发展和应用的瓶颈。克隆动物中经常出现后代过大综合征（LOS）,该病导致克隆动物早产、难产和易夭折。LOS类似于人的伯-伟综合征（BWS）,BWS也称为Wlims瘤,表现为巨舌、内脏肿大等症状。研究发现BWS的发病机理与WT1基因（Wilms’tumor 1gene）异常表达有关。本文对体细胞核重编程和表观遗传学调控细胞重编程的研究进展进行综述,并对WT1基因组蛋白乙酰化修饰与体细胞重编程之间的联系进行简要介绍,以期为生命科学领域的进一步探索与研究提供借鉴。     

Extended exposure to trichostatin A after activation alters the expression of genes important for early development in nuclear transfer murine embryos

The low viability of embryos reconstructed by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic modification errors, and reduction of those errors may improve the viability of SCNT embryos. The present study shows the effect of trichostatin A (TSA), a strong inhibitor of histone deacetylase, on the development of murine SCNT embryos. After enucleation and nuclear injection, reconstructed murine oocytes were activated with or without TSA for 6 hr (TSA-6 hr). After activation, TSA treatment was extended to 3 hr (TSA-9 hr), 5 hr (TSA-11 hr) and 18 hr (TSA-24 hr) during culture. As a result, the SCNT embryos in the TSA-11 hr group showed a remarkably higher blastocyst rate (21.1%) when compared with the nontreated embryos (3.4%), while the concentration of TSA did not significantly affect embryonic development. The expressions of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) genes decreased in the TSA-11 hr and TSA-24 hr groups, while there was an increase in the expression of histone acetyltransferase (P300 and CBP), pluripotency (OCT4 and NANOG) and embryonic growth/trophectoderm formation (FGF4)-related genes in the same groups. The expression of CDX2, a critical gene for trophectoderm formation was upregulated only in the TSA-24 hr group. Our results show that TSA treatment during the peri- and postactivation period improves the development of reconstructed murine embryos, and this observation may be explained by enhanced epigenetic modification of somatic cells caused by TSA-induced hyperacetylation, demethylation and upregulation of pluripotency and embryonic growth after SCNT.     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.