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。     

体细胞核移植与中心体遗传

体细胞克隆虽然在多种哺乳动物中成功获得后代, 但仍存在一系列的问题需要解决。克隆胚胎的发育能力由核移植后几小时内的细胞和分子过程决定, 包括染色体分离和纺锤体的重新组装。中心体的正常组成和分布能保证染色体分离的准确性及新生和出生后克隆动物发育过程中的基因组稳定性。文章在分析哺乳动物体细胞克隆存在的问题和简介中心体结构功能的基础上, 综述了中心体在配子和受精卵发育过程中的遗传机制, 同时阐述了体细胞克隆胚胎中心体及其相关蛋白的研究现状。     

克隆的概念,意义与进展

首先介绍了克隆在个体,细胞和分子水平的生物学定义,强调了克隆是一个“群体”概念。然后较为详细地讨论了分子克隆（ＤＮＡ克隆）和通过细胞核移植进行动态（小鼠）克隆的基本,意义,目前进展和有等回答的一些问题。     

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。     

供核细胞对体细胞核移植效率的影响

利用体细胞核移植技术克隆动物、生产转基因家畜具有极大的应用潜力。然而,核移植效率低下、克隆后代形态异常等问题仍然制约着体细胞核移植技术的产业化进展。影响体细胞核移植效率的因素很多,该文着重从供核细胞的类型、细胞体外培养、细胞凋亡及转基因操作等方面阐述其对体细胞核移植效率的影响。     

动物克隆的机理与研究进展

对动物克隆的理论基础进行了探讨,综述了在动物克隆时,供体核的基因组再程序化的可能作用机制,对动物克隆尤其是喷乳动物克隆研究进展进行了深入的总结和分析,并对其应用前景作了展望。     

克隆狗问世

经过许多年的失败之后，它终于诞生了。2005年8月，一组韩国科学家宣布他们首次成功地克隆出一只阿富汗猎犬。这只小狗终于加入了由绵羊、山羊、小鼠、大鼠、猫、牛、猪、马、兔子、鹿和骡子组成的克隆动物俱乐部。但是，对于一些爱狗者而言，这并不意味着让他们的宠物再生已经为期不远。     

克隆动物研究进展

本文介绍了上世界上中国条用细胞核移植技术克隆动物的研究历史。综述了细胞核移植的程序、方法和影响因素,包括受体卵母细胞的去核、供体细胞核的制备、核移植、激活、受体细胞与供体细胞的融合、重组胚的体内和体外培养以及胚胎移植产生克隆动物。对克隆动物研究和应用前景进行了讨论。近期的研究结果表明,多代克隆可产生在量遗传性相同的动物,不久的将来克隆技术在商业上的应用将成为现实。     

单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较

为比较两种筛选标记基因生产转人乳铁蛋白(hLF)基因克隆山羊的效率,利用单(新霉素抗性基因,Neor)、双(新霉素抗性和绿色荧光蛋白基因,Neor/GFP)标记基因筛选转基因的供核细胞,并制作体细胞核移植转基因山羊。山羊胎儿成纤维细胞电转染单标记基因表达载体(pBLC14)或双标记基因表达载体(pAPLM),分别有58.8%(20/34)和86.7%(26/30)的抗性细胞株检测到外源基因;转染pAPLM的细胞传代培养后,仅有20%(6/30)株细胞在传代中所有细胞均能观察到荧光;分别以pBLC14和pAPLM的细胞株作为供核细胞进行体细胞核移植,共获得806枚重构胚胎,胚胎移植受体后35 d、60 d妊娠率分别为53.8%、26.9%和39.1%、21.7%,最终分别产下5只(1.9%)和7只(1.4%)克隆山羊;经PCR及Southern blotting检测,所有出生山羊均整合有外源基因。结果显示,以单、双标记基因筛选供核细胞,其重构胚融合率、怀孕率和克隆动物出生率差异不显著(P>0.05),Neor/GFP双标记基因能准确、有效地用于转基因供核细胞筛选。同时,结果也表明Neor/GFP双标记基因转染的体细胞作为供核细胞对体细胞克隆效率未出现不利影响。     

我国体细胞克隆牛的研究进展

近年来,由于体细胞克隆技术在畜牧业生产、疾病治疗、生物学基础理论研究及濒危动物保护等诸多领域所蕴藏的巨大应用价值,克隆效率成为科学界研究的热点问题。本文对克隆技术的相关影响因素进行了综述,研究表明,不同细胞系影响到体细胞克隆牛效率,胎儿细胞和颗粒细胞重构胚的囊胚发育率、犊牛成活率比成年成纤维细胞高。关于卵母细胞和克隆胚胎冷冻保存的研究表明,玻璃化冷冻法可以用于克隆胚胎以及卵母细胞的冷冻保存。在此基础上,讨论了国际上哺乳动物体细胞克隆研究新进展和体细胞克隆的效率,指出体细胞克隆中亟待解决的问题。     

Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.     

Cloning of Asian yellow goat (C. hircus) by somatic cell nuclear transfer: telophase enucleation combined with whole cell intracytoplasmic injection

Our and other previous studies have shown that telophase enucleation is an efficient method for preparing recipient cytoplasts in nuclear transfer. Conventional methods of somatic cell nuclear transfer either by electro-fusion or direct nucleus injection have very low efficiency in animal somatic cell cloning. To simplify the manipulation procedure and increase the efficiency of somatic cell nuclear transfer, this study was designed to study in vitro and in vivo development of Asian yellow goat cloned embryos reconstructed by direct whole cell intracytoplasmic injection (WCICI) into in vitro matured oocytes enucleated at telophase II stage. Our results demonstrated that the rates of cleavage and blastocyst development of embryos reconstructed by WCICI were slightly higher than in conventional subzonal injection (SUZI) group, but no statistic difference (P > 0.05) existed between these two methods. However, the percentage of successful embryonic reconstruction in WCICI group was significantly higher than that in SUZI group (P < 0.05). After embryo transfer at 4-cell stage, the foster in both groups gave birth to offspring. Therefore, the present study suggests that the telophase ooplasm could properly reprogram the genome of somatic cells, produce Asian yellow goat cloned embryos and viable kids, and whole cell intracytoplasmic injection is an efficient protocol for goat somatic cell nuclear transfer.     

Production of cloned pigs by whole-cell intracytoplasmic microinjection

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.     

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda-rabbit interspecies nuclear transfer

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p > 0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.     

A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72-78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Development of efficient strategies for the production of genetically modified pigs

Although pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs, the efficiency of production of transgenic offspring is generally plagued by 1% of the DNA-injected embryos. Therefore, a problem with this method is the need for large numbers of pronuclear stage embryos. One great advancement would be the use of in vitro-matured (IVM) oocytes for the purpose of transgenic pig production. High developmental competence of IVM oocytes was proven by transfer of parthenogenetic IVM oocytes. A combined method of sperm vectors with the IVM of oocytes would make the production of transgenic pigs remarkably feasible. Rate of blastocyst formation following intracytoplasmic sperm injection (ICSI) by frozen sperm was over 20%, and transgene was expressed in approximately 50% of blastocysts generated. Somatic cell nuclear transfer would enable more efficient and sophisticated genetic modification of the pig. Simultaneous comparison between two nuclear transfer methods by electro-fusion and intracytoplasmic injection revealed clear differences in the pattern of nuclear remodeling and development of the reconstructed embryos. To specify the donor cell type that allows efficient genetic modification and easy reprogramming or to establish such cell lines is a critical issue in pig cloning. We tested pre-adipocytes from the subcutaneous adipose tissue of adult pigs for nuclear transfer. Cell cycle synchronization by differentiation induction is unique to the pre-adipocytes. Frequency of apoptosis was low in the cells synchronized by differentiation induction compared with other synchronization methods, including serum starvation, confluency, and chemical treatment. It would be of great worth if cryopreserved clone embryos were available. We have demonstrated that cryopreservation of in vitro-produced porcine embryos as well as clone blastocysts is possible by our unique method.     

A Comparative Study on the Efficiency of Two Enucleation Methods in Pig Somatic Cell Nuclear Transfer: Effects of the Squeezing and the Aspiration Methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.     

完整的供体细胞膜及供体胞质对小鼠体细胞克隆胚发育的影响

利用显微注射和电融合的方法都可以成功地获得体细胞克隆小鼠, 由于电融合法操作耗时, 融合率低, 因而大多数克隆小鼠是采用注射方法。而注射法需要将供体细胞核从细胞中分离出来, 此分离操作有可能导致对DNA的损伤, 曾有人使用直径较粗的注射管进行完整的供体细胞注射, 这种方法操作相对简单而且对供体核没有损伤。为了研究这种方法在小鼠核移植中是否适用, 本实验使用完整的小鼠卵丘细胞作供体, 进行显微注射, 结果显示, 完整的卵丘细胞注入卵母细胞后, 无论在1小时或者6小时激活, 大部分的重构胚在2细胞期碎裂, 而去掉细胞膜的供体体细胞核注入卵母细胞后, 重构胚可以卵裂并进一步发育。卵母细胞去核后不注射供体也发生碎裂, 大部分的孤雌胚(不去核)在完整的卵丘细胞被注入后同样发生碎裂。在供体卵丘细胞刚破膜后即被注入卵胞质和供核被充分剥离后注入两种情况下获得的重构胚的体外发育中, 前者发育各期的比率显著低于后者。这些结果说明完整的卵丘细胞膜阻碍了卵胞质对体细胞核的重编程作用, 造成碎裂; 注入卵胞质的供体质膜和胞质成分影响了克隆胚的体外发育。     

猪卵母细胞体外成熟的发育潜力及核移植重构胚构建方法的研究

为了提高猪体细胞核移植重构胚发育潜力,本研究对体外成熟28 h、32 h、36 h、40 h、44 h、48 h、52 h和56 h的猪卵母细胞分别进行去核构建重构胚.研究结果表明,成熟44 h的卵母细胞核移植后有较高的融合率(58.99%)、卵裂率(67.52%)和囊胚率(22.78%),而成熟48 h的卵母细胞则分别为56.51%、65.73%和15.96%;且卵龄为44 h的卵母细胞核移植后分裂率与囊胚率显著高于卵龄为40 h、36 h、32 h、28 h的卵母细胞的分裂率与囊胚率(P＜0.05).卵龄为48 h的卵母细胞融合率高于卵龄为52 h卵母细胞的融合率(P＜0.05).同时我们还探讨了不同去核方法(盲吸法、Hochest33342染色法和Spindle-view system)对猪体细胞核移植重构胚发育能力的影响.研究结果发现,盲吸法、Hoechest33342染色法和Spindle-view system法的去核率分别达到76.33%,100.00%和98.40%.Hoechest染色法去核率显著高于盲吸法的去核率(P＞0.05),而与Spindle-view法去核率没有差异(P＞0.05).三种方法在融合率和囊胚率方面差异不显著(P＞0.05),但Hoechest染色法的分裂率较低,差异显著(P＜0.05).进一步的研究表明,细胞质内注射进行核移植构建重构胚的分裂率和囊胚率分别为68.13%和6.44%;透明带下注射法则为60.37%和8.08%,两者差异不显著(P＜0.05);两者均可运用于猪体细胞的核移植,这为建立有效的猪体细胞核移植体系提供了参考.