鱼类核移植与重编程

鱼类核移植是动物克隆研究的一个重要领域, 我国学者在上世纪60年代首创了鱼类的核移植研究。以斑马鱼为模式动物, 进行核移植与再程序化研究具有独特的优势。文章总结了鱼类细胞核移植研究的历史、斑马鱼核移植研究概况、以及影响核移植胚胎发育的因素, 特别是核移植胚胎基因组的表观遗传修饰, 如基因组DNA甲基化及组蛋白乙酰化和甲基化等的研究, 将有助于完善克隆技术并提高克隆的成功率, 推动克隆技术的广泛开展和应用。     

各国家畜胚胎细胞核移植研究进展

细胞核移植技术就是将成熟的卵母细胞或受精卵中的细胞核除去后移植其它细胞核,使卵母细胞不经过精子穿透等有性过程就可被激活、正常分裂并发育成新的个体。因此,核移植技术可以使细胞核供体的基因通过无性繁殖手段得到完全的复制,从而产生出克隆(无性繁殖)动物。鱼类的细胞核移植的成功报道已有很多且很早。然而哺乳动物,尤其是家畜的胚胎细胞核移植是近几年才发展起来的。     

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…     

硬骨鱼类体细胞核移植的研究

本文用不同属、科、目的硬骨鱼类作材料进行体细胞核移植研究。鲫鱼（Ｃａｒａｓｓｉｕｓａｕｒａｔｕｓ）、鲮鱼（Ｃｉｒｒｈｉｎｕｓｍｏｌｉｔｏｒｅｌｌａ）和尼罗罗非鲫（Ｔｉｌａｐｉａｎｉｌｏｔｉｃａ）的体细胞核（头肾细胞）移植到鲤鱼（Ｃｙｐｒｉｎｕｓｃａｒｐｉｏ）的成熟去核卵中,通过继代核移植,在鲫鱼体细胞核和鲤鱼去核卵的属间组合中,获得发育到血液循环期的幼鱼;在鲮鱼体细胞核和鲤鱼去核卵的亚科间组合中,获得发育到心脏跳动期的晚期胚胎;在尼罗非鲫体细胞核和鲤鱼去枚卵的目间组合中,获得发育到肌肉效应期的胚胎。由于是直接用成鱼体细胞核作供核体进行核移植,因而能够克服供体鱼和受体鱼不同步产卵的困难。实验结果表明,这对进行硬骨鱼类核质杂交研究无疑是一种简便而又有效的方法。     

体细胞核移植的研究进展

自从克隆羊多利问世以后,体细胞核移植有了较大的发展,陆续有新的动物克隆成功,但克隆的效率仍然较低。本文综述了体细胞核移植技术研究的进展情况,介绍了基因背景、核移植步骤、胚胎相关支持技术对核移植成功率的影响以及核移植技术中的基因修饰和对核重新程序化的最新认识。     

外源基因在鱼类核移植胚胎中的转录起始

比较了hGH转植基因在F4代的转MThGH基因鱼, F4代胚胎细胞的核移植后代, 以及F4代尾鳍培养细胞的核移植后代中的转录时序差异. RT-PCR实验结果表明, hGH基因在转基因鱼F4代胚胎中从原肠早期开始转录; F4代胚胎细胞核移植的后代在囊胚早期已能检测到hGH基因转录本; F4代尾鳍培养细胞核移植的后代自16胞期就出现hGH基因的转录.上述结果表明, 鲤鱼卵细胞质对分化细胞核特定基因的再程序化能力是有限的, 进而推论鱼类细胞核移植试验中仅有少部分的供体核能够发生完全再程序化.     

猪胚胎细胞核移植研究的新进展

猪胚胎细胞核移植研究的新进展＠赵浩斌￥湖北省农业科学院畜牧兽医研究所猪,卵细胞,胚胎,核移植,胚胎干细胞,细胞周期猪胚胎细胞核移植研究的新进展赵浩斌（湖北省农业科学院畜牧兽医研究所武汉４３０２０９）关键词猪卵细胞胚胎核移植胚胎干细胞细胞周期核移植就是将供...     

哺乳动物体细胞核移植研究进展

体细胞核移植技术已经在基础研究领域与产业化应用领域体现出了重要的价值,因而体细胞核移植技术及其相关研究已经成为了生物领域的持续性研究热点,但是围绕体细胞核移植技术仍然存在许多质疑,其中最主要的就是体细胞核移植的效率较低。尽管如此,体细胞核移植研究仍然在近年来取得了令人瞩目的成就,包括小鼠与恒河猴核移植胚胎干细胞系的建立。该文就体细胞核移植的研究历史与进展进行简要的论述,同时针对体细胞核移植研究中的细胞重编程与治疗性克隆研究中的发展与问题进行剖析,希望能够积极推动治疗性克隆的研究进展,加速核移植与干细胞技术在产业化领域中的应用。     

猪体细胞核移植的研究进展和影响因素

自2000年Polejaeva IA获得第1头克隆猪后,短短几年时间全世界已有10多例成功的报道,使得猪的体细胞核移植有了长足的发展,但目前猪的体细胞核移植效率依然低下（1—2％）,人们对核移植中重编程分子机理的认识知之甚少。简要综述了猪体细胞核移植近年来的研究进展,就猪核移植中的技术难点和影响因素进行了分析,涉及供体细胞种类的选择、体外长期培养和高压筛选对随后核移植的影响以及供核细胞细胞周期的选择,核质双方的协调,去核和注核方法的选择,融合和激活程序的优化,妊娠的维持等。     

动物异种细胞核移植技术研究进展

作为研究动物胚胎发育过程中核质关系重要手段的细胞核移植技术正在逐步完善。异种细胞核移植技术不仅可用于保护濒危野生动物和珍贵的动物遗传资源,而且在研究核质相互作用和物种之间的进化关系上有着独特的用途。尽管目前同种核移植在许多哺乳动物上都获得成功,但异种核移植研究还刚刚开始,有许多问题亟待解决。本仅就现已发表的少量献,简要综述了动物异种核移植研究的历史与现状、影响因素、面临问题和应用前景。     

小鼠体细胞核移植程序的研究

采用直接去核法、透明带切割法、PMM法3种核移植方法进行小鼠卵母细胞去核的研究。3种方法都未对卵母细胞核进行示踪,仅以第一极体作为参照进行去核,都属于盲吸法范畴,在去核率上没有显著差异。直接去核法对卵母细胞造成较大的伤害,去核操作过程中极易使卵母细胞膜破裂,发生崩解;透明带切割法分步操作使操作变得柔和,对卵母细胞的刺激减小,去核卵母细胞的存活率较高;PMM法靠脉冲电压在透明带上打孔进行辅助去核,脉冲参数稍大或压透明带太紧都极易在击破透明带的同时击破卵膜,使卵母细胞发生崩解。在构建重构胚的过程中,胞质内注射法较电融合法而言,程序简单,带入的供体胞质较少,构建重构胚的效率更高。     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.     

Mouse cloned from embryonic stem (ES) cells synchronized in metaphase with nocodazole

Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001.     

Nuclear transplantation in the pig embryo: nuclear swelling

The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase II oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4-, 8-, and 16-cell stage embryos swelled to a similar diameter after nuclear transfer (26.9, 27.3, and 27.2 microns, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3, and 13.0 microns, respectively). This is a swelling of 47, 91, and 109%, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract

We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein) and two activation times (1.5-2 vs. 8-10 h after nuclear transfer) were examined. Cleavage and activation (pseudopronucleus formation) rates of oocytes injected with sperm extract containing 59 microg/ml protein were significantly (P < 0.05) lower than any other dosage. The percentage of embryos cleaving with normal nuclei in oocytes injected with the 1375 microg/ml preparation 1.5-2 h after donor injection was significantly (P < 0.05) higher than that of the 293 microg/ml preparation 8-10 h after donor injection (22 vs. 6%). Embryos developed to a maximum of 10 nuclei. Interspecies nuclear transfer was performed by direct injection of horse nuclei into enucleated bovine oocytes, followed by chemical activation. This resulted in 81% reconstruction (successful injection of the donor cell), 88% cleavage, and 73% cleavage with normal nuclei. These results indicate that direct nuclear injection using the Piezo drill is an efficient method for nuclear transfer in horse and cattle oocytes and that sperm extract can efficiently activate horse oocytes both parthenogenetically and after nuclear transfer     

Cloned calves from chromatin remodeled in vitro

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.     

Nuclear transplantation as a method of producing genetically identical livestock

Abstract

Genetic variation is a problem faced by both researchers and producers. One method to reduce genetic variation is to clone embryos by nuclear transfer. Implementation, involves transferring nuclei from a morula stage embryo to unfertilized oocytes from which the metaphase II chromosomes have been removed. Since each of the nuclei from the original morula stage embryo are genetically identical, each of the embryos that are a result of nuclear transfer have identical nuclear genetics. The original morula stage embryo, relatively speaking, is more differentiated than a one‐cell stage embryo. Thus for the resulting nuclear transfer embryo to continue in development the transferred nuclei must be remodeled to resemble nuclei of a one‐cell stage embryo and be reprogrammed in their developmental cascade of events to behave as nuclei of a one‐cell stage embryo. The potential applications of producing genetically identical individuals range from reducing the number of animals needed for experimentation to providing a more uniform product in the freezer at the grocery store. Unfortunately, the procedures for producing cloned animals by nuclear transfer are still relatively inefficient. There is a need for more basic research to be conducted in understanding mammalian embryogenesis for the application of this and other biotechnologies.