不同活化方法对小鼠卵母细胞孤雌发育的影响(简报)

随着动物克隆技术的不断发展,核移植胚胎的活化成为一个很重要的研究领域,活化的效率直接影响到克隆的成功率。小鼠体细胞克隆研究大多采用卵胞质直接注射供体细胞核的方法构建重构胚,再经SrCl_2活化处理进行体外发育。也有经电融合,SrCl_2活化产仔的报道。已有研究表明单独SrCl_2处理或电刺激均能很好地活化小鼠卵母细胞,那么在小鼠核移植过程中采用的电融合条件是否能激活卵母细胞,电脉冲刺激后再经SrCl_2处理是否能     

哺乳动物体细胞克隆技术的研究进展

尽管与已有十几年发展史的哺乳动物胚胎细胞克隆技术相比,其体细胞克隆技术的研究仅有不到三年的时间;然而,鉴于哺乳动物体细胞克隆技术在供核效率等方面所具备的明显优势,其应用价值远高于胚胎细胞克隆技术。随着对哺乳动物体细胞克隆技术基本机理的进一步广泛深入的研究,它将在大量克隆优良种畜、扩大同基因型实验动物种群、培育转基因动物和保护濒危野生动物遗传资源等方面发挥巨大作用。     

哺乳动物体细胞克隆技术的研究进展

尽管与已有十几年发展史的哺乳动物胚胎细胞克隆技术相比,其体细胞克隆技术的研究仅有不到三年的时间;然而,鉴于哺乳动物体细胞克隆技术在供核效率等方面所具备的明显优势,其应用价值远高于胚胎细胞克隆技术。随着对哺乳动物体细胞克隆技术基本机理的进一步广泛深入的研究,它将在大量克隆优良种畜、扩大同基因型实验动物种群、培育转基因动物和保护濒危野生动物遗传资源等方面发挥巨大作用。     

Leptin对陕北白绒山羊体细胞核移植的影响

Leptin的功能较为复杂,主要调控机体能量代谢。有研究表明Leptin在卵母细胞成熟及胚胎发育过程中也具有重要作用。本研究在卵母细胞体外成熟基础培养液中添加了不同浓度的Leptin,其中未添加Leptin的设为Ⅰ组,添加10ng/mL和50ng/mL Leptin的分别设为Ⅱ组和Ⅲ组。以陕北白绒山羊皮肤成纤维细胞为供体细胞,三组体外培养成熟的卵母细胞作为受体,利用显微操作方法对成熟卵母细胞进行去核操作,然后将供体细胞注射到卵周隙内,经电融合后形成体细胞克隆胚。根据卵母细胞体外成熟率、核移植效率以及克隆胚囊胚发育率分析Leptin在山羊核移植中的作用。结果表明Ⅰ组山羊卵母细胞体外成熟率和核移植效率显著高于其他两组(P<0.05),三组克隆囊胚发育率无显著差异(P>0.05)。Leptin降低了山羊卵母细胞体外成熟和核移植效率,对克隆胚发育能力无影响。     

家畜体细胞克隆中核重编程机理研究进展

体细胞克隆在绵羊、山羊、牛、猪等家畜中获得了成功,但目前的克隆效率非常低。克隆效率低使家畜体细胞克隆技术在畜牧业生产及其他领域的应用受到极大的限制,问题的根源在于对体细胞克隆中核重编程的分子机理缺乏了解。供体细胞核移入去核的卵母细胞后,必须经过后成表观遗传修饰的重编程,从而恢复供体细胞核的全能性,才能保证重构胚的正常发育及个体的正常生长。本文从移植核的重构、DNA甲基化总体改变、组蛋白修饰、X染色体失活、端粒长度和端粒酶活性恢复、印迹基因及其他与发育相关基因的表达及核重编程的影响因素等几个方面探讨了体细胞克隆中的核重编程机理,为克隆效率提高的方法研究提供理论依据。     

体细胞克隆牛产品安全分析

体细胞克隆技术是将已分化的体细胞移到去核的成熟卵母细胞中,通过体外激活和培养,再移植入受体母畜子宫内,繁殖出具有相同基因型后代的一种技术。该技术可以大幅提升繁殖效率,并提供高质、充足和营养丰富的动物食品。近年来,美国、日本和欧洲等国家相继宣布体细胞克隆动物食品可以上市。然而,目前体细胞克隆效率相当低下,即使是出生的克隆动物也往往伴随发育畸形或高死亡率等现象,在对克隆动物发育异常知之甚少的情况下,宣布克隆动物产品上市是否为时过早?以下综述了克隆牛肉、奶及其产品安全。     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。     

奶牛体细胞核移植胚胎产业化生产条件优化北大核心CSCD

通过优化高产奶牛体细胞克隆胚胎体外生产技术条件,制备高质量的奶牛克隆胚胎,旨在提高奶牛体细胞核移植产业化应用效率。就受体卵母细胞去核方法、不同年龄供体牛细胞来源、血清饥饿与否以及不同气相组成培养等条件对奶牛体细胞克隆胚胎生产效率的影响进行了研究和探讨。结果表明,虽然荧光染色辅助去核和盲吸法的去核率、囊胚发育率分别为100%、24.83%和92.44%、28.26%,两者之间无显著差异(P>0.05),但盲吸法操作简单、效率高;不同年龄来源供体牛的细胞系构建的克隆胚胎的囊胚发育率分别为31.43%、25.68%,两者之间没有显著差异(P>0.05);经血清饥饿和未饥饿供体细胞重构的克隆胚胎囊胚发育率分别为24%、29.9%,两者之间没有显著差(P>0.05);富氧和低氧气相培养的克隆胚胎的囊胚发育率分别为28.26%、31.55%,两者之间差异不显著(P>0.05),低氧气相组成更有利于囊胚的发育。根据上述结果,奶牛体细胞核移植胚胎(克隆胚胎)的产业化生产条件为:供体细胞无需进行同期饥饿处理,直接注入到盲吸去核后的受体卵子透明带下构建克隆胚胎,融合后的克隆胚胎在密封的混合三气(5%CO2-5%O2-90%N2)的气相组成下进行体外培养,能保持稳定的囊胚发育率。     

化学诱导法——卵母细胞去核新策略

哺乳动物体细胞克隆技术在过去的几年里取得了飞速发展 ,但是核移植的效率依然很低。于是人们不断的从各个方面进行探索 ,去核方法随之也成为其中的一个热点。但是传统的物理去核存在着技术要求高、耗费时间长、对细胞损伤大的缺点 ,作为思路转换的产物 ,一种新的卵母细胞去核技术———Deme诱导去核引起了各国科学家的广泛关注。文章着重介绍了Deme诱导去核辅助的核移植程序 ,Deme诱导去核成功率的影响因素 ,Deme诱导去核方法对卵母细胞及克隆胚胎的影响 ,并结合作者从事的研究对该方法目前存在的问题及某些环节可能的改进措施提出了看法。无论如何 ,Deme诱导去核方法的有效应用仍然需要作进一步的深入研究。     

核移植技术研究进展

核移植(nuelear transplation,NT)是将动物早期胚胎或体细胞的细胞核移植到去核的受精卵或成熟卵母细胞中、重新构建新的胚胎,使重构胚发育为与供核细胞基因型相同后代的技术过程,又称动物克隆技术。广义的胚胎克隆技术还包括胚胎分割和卵裂球培养,通常所指的胚胎克隆技术是指狭义概念,即核移植技术。1938年Spmann在所有胚胎细胞都具有与受精卵完全相同、拥有潜在发育全能性的细胞核基础上提出了细胞核移植的概念[‘1。早期核移植实验是在变形虫、蛙、爪蟾、非洲爪蛙等两栖类和鱼类上进行的核质关系研究〔“一“〕,随着胚胎技术的不断进步…     

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

Cloning the laboratory mouse.

A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two–cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1*b/ of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?     

Interspecies nuclear transfer of Tibetan antelope using caprine oocyte as recipient

Interspecies nuclear transfer is an invalulable tool for studying nucleus-cytoplasm interactions; and at the same time, it provides a possible alternative to clone endangered animals whose oocytes are difficult to obtain. In the present study, we investigated the possibility of cloning Tibetan antelope embryos using abattoir-derived caprine oocytes as recipients. Effects of culture conditions, enucleation timing, and donor cell passages on the in vitro development of Tibetan antelope-goat cloned embryos were studied. Maternal to zygotic transition timing of interspecies Tibetan antelope embryos was also investigated using two types of cloned embryos, Tibetan antelope-rabbit and Tibetan antelope-goat embryos. Our results indicate that: (1) goat oocyte is able to reprogram somatic cells of different genus and supports development to blastocyst in vitro. (2) Coculture system supported the development of Tibetan antelope-goat embryos to blastocyst rate stage (4.0%), while CR1aa alone did not. (3) When MII phase enucleated caprine cytoplast and TII phase enucleated caprine cytoplast were used as recipients, the fusion rate and blastocyst rate of hybrid embryos were not statistically different (73.9% vs. 67.4%; 4.0% vs. 1.1%). (4) When donor cells at 3-8 passages were used, 2.9% hybrid embryos developed to blastocysts, while none developed to blastocysts when cells at 10-17 passages were used. (5) There may be a morula-to-blastocyst block for Tibetan antelope-goat, while there may be an 8- to 16-cell block for Tibetan antelope-rabbit embryos.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

Two-staged nuclear transfer can enhance the developmental ability of goat�Csheep interspecies nuclear transfer embryos in vitro

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.     

Cloned mice derived from somatic cell nuclei

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.     

Asymmetric nuclear reprogramming in somatic cell nuclear transfer?

Despite the progress achieved over the last decade after the birth of the first cloned mammal, the efficiency of reproductive cloning remains invariably low. However, research aiming at the use of nuclear transfer for the production of patient-tailored stem cells for cell/tissue therapy is progressing rapidly. Yet, reproductive cloning has many potential implications for animal breeding, transgenic research and the conservation of endangered species. In this article we suggest that the changes in the epi-/genotype observed in cloned embryos arise from unbalanced nuclear reprogramming between parental chromosomes. It is probable that the oocyte reprogramming machinery, devised for resident chromosomes, cannot target the paternal alleles of somatic cells. We, therefore, suggest that a reasonable approach to balance this asymmetry in nuclear reprogramming might involve the transient expression in donor cells of chromatin remodelling proteins, which are physiologically expressed during spermatogenesis, in order to induce a male-specific chromatin organisation in the somatic cells before nuclear transfer.