哺乳动物克隆技术及其研究进展

综述了哺乳动物克隆技术的现状和发展趋势。介绍了哺乳动物胚胎细胞克隆、体细胞克隆技术和转基因动物克隆技术的研究进展以及我国哺乳动物克隆技术发展情况。     

哺乳动物克隆技术的研究进展

本文针对近年来的哺乳动物克隆技术的研究热点,分别从体细胞克隆方法,克隆动物的细胞核重编程,异种克隆及其治疗性克隆等方面进行了分析,并且对克隆技术在转基因中的应用进行了总结。     

哺乳动物克隆技术的研究进展

本文针对近年来的哺乳动物克隆技术的研究热点,分别从体细胞克隆方法,克隆动物的细胞核重编程,异种克隆及治疗性克隆等方面进行了分析,最后就克隆技术在转基因中的应用进行了总结。     

哺乳动物体细胞克隆研究进展

哺乳动物体细胞克隆技术是近年来才发展起来的技术，但体细胞克隆绵羊－多利的诞生却引起了世界轰动，充分显示了其重在的科学研究价值和潜在的应用价值。本文对哺乳动物体细胞克隆技术研究现状，理论基础研究，应用等方面作一综述。     

动物克隆技术研究的历史、现状与展望

动物克隆技术对于优良种畜的复制、减少实验用动物数目、动物遗传多样性保存及濒危动物挽救、转基因动物培育以及人类疾病的治疗都具有重要的意义。尽管早在1938年HansSpemann就已提出了胚胎细胞核移植的设想,但直到20世纪50和60年代才在两栖类和鱼类获得成功。而哺乳动物的胚胎细胞核移植起步更晚,直到20世纪80年代才开始有成功的报道。自1997年Wilmut等人培育出体细胞克隆绵羊Dolly以来,哺乳动物克隆技术有了迅速发展;先后获得了体细胞克隆小鼠、牛、山羊、猪、猫和兔,供核细胞的种类也在不断拓宽。然而,目前体细胞克隆的成功率还很低,一般只有1%～3%。一般认为,造成克隆成功率低的原因主要是供体核后成性基因程序再编不当。尽管最近的实验证明,端粒长度和X-染色体灭活等程序再编正常,但于配子发生期间形成的后成性信息(如配子基因印迹)在核移植之后并不能恢复。     

马体细胞克隆技术研究进展

哺乳动物体细胞克隆技术是近年来发展起来的高新技术，体细胞克隆绵羊——多利的诞生已引起了世界的轰动，笔者就关于马的体细胞克隆技术、重组卵母细胞的体外构建、激活、培养和应用前景等作一综述。     

哺乳动物体细胞克隆技术研究进展

哺乳动物体细胞克隆技术又称哺乳动物体细胞核移植技术或无性繁殖技术，它是通过特殊的人工手段（显微操作、电融合等），对哺乳动物特定发育阶段的核供体（体细胞核）及相应的核受体（去核的原核胚或成熟的卵母细胞），不经过有性繁殖过程，进行体外重构，并通过重构胚的胚胎移植，从而达到扩繁同基因型哺乳动物种群的目的。目前，用核移植技术已生产出了几种体细胞克隆哺乳动物。体细胞动物在畜牧业实际生产中，哺乳动物体细胞克隆技术的成功使快速、大量生产优良种畜成为可能。在医学上，体细胞克隆技术为生产成年个体的胚胎干细胞、复制…     

动物体细胞克隆技术的研究进展

体细胞克隆绵羊“多莉”(Dolly)的诞生,表明成年哺乳动物体细胞具有基因组的全能性。本文从“多莉”诞生的背景、体细胞克隆技术的研究现状、体细胞核移植过程中的核质互作、该技术目前存在的问题及其在制作转基因动物上的应用等方面,概述了体细胞克隆技术的研究进展。     

动物体细胞克隆技术的研究进展

体细胞克隆绵羊“多莉”（Dolly）的诞生,表明成年哺乳动物体细胞具有基因组的全能性。本文从“多莉”诞生的背景、体细胞克隆技术的研究现状、体细胞核移植过程中的核质互作、该技术目前存在的问题及其在制作转基因动物上的应用等方面,概述了体细胞克隆技术的研究进展。     

核移植克隆哺乳动物的研究进展

对核移植的基本操作程序、细胞周期及受体卵质重编程序能力,对核移植克隆哺乳动物效率的影响,以及哺乳动物体细胞克隆技术进展进行了系统综述。     

延边黄牛体细胞核移植的研究进展

牛体细胞核移植(somatic cell nuclear transfer,SCNT)是一套极其复杂的技术体系,它包括卵母细胞的成熟、供核细胞的准备、卵母细胞的显微操作、细胞融合、卵母细胞激活和胚胎培养。因此,许多因素影响着核移植胚胎发育。虽然陆续有克隆牛出生的报道,但是克隆效率依然低下。本综述对延边黄牛体细胞核移植体系的6个方面进行简单综述,以期为探究延边黄牛体细胞克隆的最优化条件、建立最优化的培养体系、提高克隆效率提供参考。     

Research Advances on Somatic Cell Nuclear Transfer in Yanbian Yellow Cattle

Bovine somatic cell nuclear transfer (SCNT) is a sophisticated technique system,including oocyte maturation,donor cell preparation and oocytes microscopic operation,fusion,activation and culture.Although the birth of cloning cattle has been reported recently,the efficiency of somatic cell cloning has remained lowly.In order to establish the optimization somatic cell nuclear transfer system of Yanbian Yellow cattle,this review summarized only from 6 main aspects mentioned above in this field.     

Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.     

Developmental competence of HMC(TM) derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells

To enable us to handle a large number of oocytes at a given time and to have an increased throughput of cloned embryos, we attempted the Handmade cloning (HMC) technique, a zona-free method of bovine somatic cell nuclear transfer. Our objective was to study the developmental competence of the HMC derived embryos obtained using different types of somatic cells. A total of 6,874 cumulus-oocyte-complexes were used with either 7th or 11th passage fibroblasts (1st and 2nd groups, respectively), which were prepared from male animals, or granulosa cells (3rd group) as nuclei donors. The average cleavage rate was 65%, accompanied by a blastocyst rate of just 2% for the cleaved products and 5% for the >8-cell embryos, and there was no significant difference between the three groups. Out of 27 blastocysts recovered, 22 blastocysts were transferred to 22 recipients, resulting in two pregnancies. One pregnancy was lost after the fourth week while the other progressed to full term with the birth of a male calf. This first successful cloning of a male calf with the HMC technique in Europe indicates the successful adoption and establishment of this technique in our laboratory, and that this technique can be successful in producing viable embryos.     

哺乳动物的克隆技术——哺乳动物克隆的原理、方法、影响因素及存在的问题

哺乳动物细胞克隆是20世纪末生命科学领域最引人注目的高新技术,该技术对于优良种畜的复制、减少试验用动物数目、动物遗传多样性保存及濒危动物挽救、转基因动物培育等方面具有重要意义。近年来克隆技术发展迅速,多种哺乳动物相继克隆成功,但也存在克隆效率太低、克隆动物表型正常而实质异常的问题。作者阐述了动物克隆的基本原理、操作方法及其相关影响因素,提出了解决相关问题的关键。     

动物体细胞克隆研究概况

介绍了动物体细胞克隆技术的方法、研究现状，指出体细胞克隆动物虽然存在成功率低、死亡率高、生长发育不正常等缺陷，但在快速扩繁优良种畜和转基因动物、保护动物遗传资源、生产用于器官移植的克隆器官等方面有很大的应用潜力。     

Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.     

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.     

中药添加剂降低牛奶中体细胞作用的功效试验

乳汁中体细胞增高,是奶牛隐性乳腺炎的主要指标,用传统中兽医理论组方,针对奶牛隐性乳腺炎,配制了一种具有清热解毒、通经活络和提高乳房抵抗力及免疫功能的中药添加剂——"降体细胞中药添加剂",并对奶牛隐性乳腺炎病例进行了降低体细胞的治疗试验。结果显示,其一个疗程(7d)对奶牛隐性乳腺炎病例降低体细胞功能的有效率为83%,略高于市售产品对照组80%的有效率。因为是纯中药配方,可作为市售同功效但泌乳期禁用药物的替代产品。