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胚胎细胞克隆猴流程Cloning of primate embryos has been a topic of much debate and controversy within the scientific community. The process of cloning monkeys involves a series of intricate steps that require careful precision and expertise. Scientists are able to clone monkeys by taking the nucleus of a somatic cell from an adult monkey and transferring it into an egg cell that has had its nucleus removed. This reconstructed egg is then stimulated to begin dividing and developing into an embryo, which can then be implanted into a surrogate mother.胚胎细胞克隆猴是科学界内一个备受争议和争论的话题。
克隆猴的过程涉及一系列复杂的步骤，需要仔细的精密和专业知识。
科学家可以通过从成年猴的体细胞中取出细胞核，将其转移到已经取出细胞核的卵细胞中来克隆猴。
然后需要刺激这个重组卵细胞开始分裂并发育成一个胚胎，然后将其植入代孕母体。
The process of cloning primate embryos is a complex one, involving a number of technical challenges that must be overcome. One such challenge is the low success rate of cloning in primates, with manyembryos failing to develop or resulting in genetic abnormalities. This low success rate can be attributed to the difficulty of manipulating primate eggs and embryos compared to other species. Additionally, the process of cloning primates is ethically contentious, with concerns about the welfare of the cloned animals and the implications for human cloning.胚胎细胞克隆猴的过程是一个复杂的过程，涉及许多必须克服的技术挑战。
胚胎移植牛英语作文Title: Embryo Transfer in Cattle。
Embryo transfer in cattle, a widely practiced reproductive technique in the field of animal husbandry, plays a pivotal role in enhancing genetic progress, accelerating breeding programs, and conserving valuable genetics. In this essay, we will delve into the process of embryo transfer, its significance, and its impact on the cattle industry.To begin with, embryo transfer involves the removal of embryos from a genetically superior donor female and their subsequent transfer into recipient females that serve as surrogate mothers. This technique allows breeders to amplify the reproductive potential of elite females, thereby maximizing the dissemination of desirable genetic traits within a population.The process of embryo transfer typically comprisesseveral key steps. Firstly, superovulation is induced inthe donor female through hormone treatments to stimulatethe production of multiple ova. Subsequently, the donor female undergoes artificial insemination with semen from a carefully selected bull possessing desirable genetic traits. Following insemination, embryos are collected from the reproductive tract of the donor female via non-surgical or surgical methods. These embryos are then evaluated for quality and developmental stage before being transferredinto synchronized recipient females. The recipient females are closely monitored to ensure successful implantation and pregnancy establishment.The significance of embryo transfer in cattle breeding cannot be overstated. One of its primary benefits is the acceleration of genetic progress within a population. By concentrating the genetic contributions of superior individuals, embryo transfer facilitates the rapid dissemination of desirable traits such as milk production, meat quality, disease resistance, and reproductive efficiency. Moreover, this technique allows breeders to overcome logistical constraints associated with naturalmating, such as geographical distances and reproductive incompatibilities between animals. As a result, breeders can access genetic material from elite individuals regardless of their location, thereby expanding the genetic base of their herds and increasing the overall competitiveness of their breeding programs.Furthermore, embryo transfer contributes to the conservation of valuable genetics and the preservation of endangered or rare breeds. By capturing and preserving the genetic material of exceptional individuals, breeders can safeguard against the loss of genetic diversity and prevent the extinction of valuable livestock populations. This is particularly crucial in the context of heritage breeds or species with limited population sizes, where genetic variability is essential for long-term sustainability.In addition to its practical applications, embryo transfer also presents ethical considerations that warrant careful attention. Critics argue that the manipulation of reproductive processes may compromise animal welfare by subjecting animals to invasive procedures and hormonaltreatments. Furthermore, concerns have been raised regarding the commodification of animal genetics and the potential for exploitation of animals as mere breeding machines. It is imperative for breeders and researchers to uphold high ethical standards and prioritize the welfare of animals throughout the embryo transfer process.In conclusion, embryo transfer represents a powerful tool for enhancing genetic progress, accelerating breeding programs, and conserving valuable genetics in the cattle industry. While its benefits are undeniable, it isessential to approach this technique with a commitment to ethical principles and animal welfare. By leveraging the potential of embryo transfer responsibly, breeders can contribute to the sustainable advancement of livestock production and the preservation of genetic diversity for future generations.。
克隆羊多莉英语作文The Story of Dolly: The Breakthrough of Cloning Technology.In the annals of scientific history, few achievements have captivated the imagination and sparked debate as much as the cloning of the sheep, Dolly. This remarkable feat marked a milestone in biotechnology, raising questionsabout the ethical, medical, and philosophical implicationsof human cloning. Dolly's birth in 1996 was the culmination of years of research and experimentation, and her very existence challenged our understanding of what was possible.The concept of cloning, or replicating an organism exactly, had been a subject of scientific speculation for centuries. However, it was only in the late 20th centurythat the technological advancements necessary to make thisa reality began to emerge. The key to Dolly's creation layin a process called somatic cell nuclear transfer (SCNT). This involved removing the nucleus from an egg cell andreplacing it with the nucleus of a somatic cell (a cellfrom the body that is not a sex cell). The egg cell wasthen stimulated to divide and develop into an embryo, which could then be implanted into a surrogate mother to carry to term.The creation of Dolly was led by a team of British researchers at the Roslin Institute, led by scientist Ian Wilmut. They began their journey with the simple goal of understanding how to control the development of embryos in vitro. However, their experiments soon led them to thebrink of a scientific revolution. After numerous failed attempts and setbacks, they succeeded in creating Dolly using a udder cell from a six-year-old sheep as the sourceof the nucleus. Dolly's birth on July 5, 1996, marked the first time that a mammal had been cloned from an adult cell.The announcement of Dolly's birth caused a stir in the scientific community and beyond. It sparked debates about the ethics of cloning, particularly in relation to human cloning. Many questioned whether it was morally acceptableto create copies of human beings or to use cloning forreproductive purposes. Others pointed to the potential medical benefits of cloning, such as the possibility of creating organs for transplantation or treating genetic diseases.The scientific implications of Dolly's cloning were also profound. Her birth demonstrated that the aging process was not entirely determined by genes, as previously thought, but could also be influenced by the environment and the way cells interact. This had the potential to revolutionize our understanding of aging and disease.Moreover, Dolly's creation highlighted the incredible power of stem cells. By using adult cells to create Dolly, the researchers effectively reprogrammed these cells back to an embryonic state, where they could divide and develop into any type of cell in the body. This offered new hopefor regenerative medicine and the treatment of a wide range of diseases.However, Dolly's life was not without its challenges. She suffered from arthritis and other health issues, whichsome attributed to the cloning process. She also died at a relatively young age, just six years old, due to lung disease. These issues raised concerns about the long-term health effects of cloning and the welfare of cloned animals.Despite these challenges, Dolly's legacy remains. She has become a symbol of scientific achievement and acatalyst for further research in cloning and biotechnology. Her story has inspired scientists to push the boundaries of what is possible, while also prompting us to consider the ethical and moral implications of our scientific pursuits. Dolly's impact on science and society is immeasurable, and her story will undoubtedly continue to inspire andchallenge us for generations to come.。
关于克隆动物的英文作文英文：Cloning animals has been a controversial topic for many years. On one hand, some people believe that cloning can bring many benefits, such as producing healthier and more productive animals for agriculture or even cloning endangered species to prevent extinction. On the other hand, others argue that cloning is unethical and can havenegative effects on animal welfare.Personally, I believe that cloning animals is a complex issue and there are both pros and cons to consider. For example, cloning can be beneficial in the case of endangered species. By cloning these animals, we canprevent them from going extinct and preserve biodiversity. However, cloning can also have negative effects on animal welfare, as cloned animals often suffer from healthproblems and have a lower life expectancy.Another issue to consider is the cost of cloning. Cloning is an expensive process and it may not be economically feasible for many farmers or conservation organizations. In addition, there is also the issue of genetic diversity. By cloning animals, we are essentially creating genetic copies, which can lead to a lack ofgenetic diversity and increase the risk of diseases andother problems.In conclusion, cloning animals is a complex issue that requires careful consideration. While there are potential benefits to cloning, such as preserving endangered species, there are also significant drawbacks to consider. Ultimately, we must weigh the pros and cons and make informed decisions about whether or not to clone animals.中文：克隆动物长期以来一直是一个有争议的话题。
猪的基因序列英语作文Title: The Genomic Blueprint of Pigs: Unraveling the Genetic Sequence。
Pigs, as essential livestock animals, have been a cornerstone of human civilization for thousands of years. Their genetic makeup holds the key to understanding various aspects of their biology, evolution, and potential applications in agriculture and biomedicine. In this essay, we delve into the intricate world of pig genomics, exploring the significance of their genetic sequence andits implications.Introduction to Pig Genomics:The field of genomics, particularly pig genomics, encompasses the study of the entire genetic material of pigs, including their DNA sequence, gene structure, and function. The completion of the pig genome sequencing project in 2012 marked a significant milestone inunderstanding the genetic blueprint of these animals. With approximately 2.7 billion base pairs, the pig genome is comparable in size to the human genome, comprising a complex network of genes and regulatory elements.Genetic Diversity and Evolution:The genetic diversity among pig breeds is extensive, reflecting centuries of selective breeding by humans for various traits such as meat quality, disease resistance, and reproductive performance. By analyzing the genetic variation present within and between different pig populations, researchers can reconstruct the evolutionary history of pigs and uncover the genetic basis of traitsthat have been shaped by natural selection and artificial selection.Functional Genomics:Functional genomics aims to decipher the functions of genes and their interactions within biological systems. Through techniques such as transcriptomics, proteomics, andmetabolomics, scientists can elucidate how genes are expressed, regulated, and ultimately contribute to the phenotype of an organism. In pigs, functional genomics studies have provided insights into important biological processes such as growth, development, immunity, and reproduction.Applications in Agriculture:The knowledge gained from pig genomics has practical implications for agricultural practices aimed at improving pig breeding, production efficiency, and disease management. Genomic selection, for instance, allows breeders toidentify superior individuals for breeding based on their genetic potential for desirable traits, leading to accelerated genetic progress and increased productivity. Additionally, genomic technologies enable the detection of genetic markers associated with disease resistance,enabling more targeted breeding strategies to mitigate the impact of infectious diseases in pig populations.Biomedical Relevance:Beyond agriculture, pig genomics has relevance in biomedical research, particularly in the field of comparative genomics and xenotransplantation. Pigs share physiological and anatomical similarities with humans, making them valuable models for studying human diseases and developing novel therapies. By editing pig genomes using advanced gene editing techniques such as CRISPR-Cas9, researchers can generate pig models with genetic modifications that mimic human disease conditions, facilitating the development of new treatments and therapies.Challenges and Future Directions:Despite the significant progress in pig genomics, several challenges remain, including the identification of functional elements within the genome, understanding the complex regulatory networks that govern gene expression, and addressing ethical considerations associated with genetic manipulation in pigs. Future research efforts will likely focus on integrating genomic data with other omicsdata sets, developing innovative breeding strategies, and advancing genome editing technologies to further harness the potential of pig genomics in agriculture, medicine, and beyond.Conclusion:In conclusion, the genomic sequence of pigs holds immense promise for advancing our understanding of pig biology, evolution, and applications in agriculture and biomedicine. By unraveling the complexities of the pig genome, scientists can unlock new opportunities for enhancing pig breeding, improving production efficiency, and addressing global challenges such as food security and human health. The journey of exploration into pig genomics continues to evolve, promising exciting discoveries and innovations in the years to come.。
Germany embryologists pointed out for the first time, and then was born dolly, then what the pig sheep cow came,Use the way monkey cloning embryos split researchers recently announced that they in biotechnology fields have achieved a landmark progress. Scientists use the cloning of embryos split ways, created a monkey. Rather, it is a rhesus monkeys, named "TaiTeLa", it is the first time scientists have been cloned way to foster primates.Now the technology have been developed to human cloning is not a problem, but it is the condemnation of the factors against, so it YuKeLong application on organs necrosis, for the benefit of mankind.德国胚胎学家首次提出的，然后就诞生了克隆羊多莉，接着的什么猪羊牛的都来了，运用分裂胚胎的方式克隆猴子科研人员最近宣布,他们在生物工艺学领域取得了一项有里程碑意义的进展。
科学家们利用分裂胚胎的克隆方式,培育出了一只猴子。
确切地说,这是一只恒河猴,名叫“泰特拉”,它是科学家首次以克隆方式培育出的灵长目动物。
现在的技术已经发展到克隆人是不成问题的，但这会受到社会的谴责的因素的反对，所以就把它应用于克隆坏死的器官上，为人类造福。
·研究论文·Chinese Journal of Animal Infectious Diseases中国动物传染病学报猪TRIM32分子的克隆、表达及其功能分析摘 要：TRIM32作为TRIM 家族中的一员，在宿主先天性免疫防御中起着重要的作用。
然而，现今对猪TRIM32分子的研究鲜有报道。
首先，利用RT-PCR 从猪肺中扩增获得了猪TRIM32分子，并将其克隆入真核表达载体p3xFlag-CMV-14，构建重组真核表达载体pFlag-porTRIM32。
随后，在Marc145细胞上利用过表达猪TRIM32，鉴定其在PRRSV 感染中的作用。
此外，为了进一步验证其在PRRSV 感染中的作用，在沉默Marc145细胞中内源性TRIM32表达的情况下，检测了PRRSV 的感染情况。
结果显示：成功构建了pFlag-porTRIM32真核表达载体；Western blot 的结果显示，Flag 抗体能识别瞬时表达的融合蛋白Flag-porTRIM32；在Marc145细胞上过表达猪TRIM32分子，可显著地抑制PRRSV 的复制；相反，在Marc145细胞上干扰TRIM32的表达，可明显增加PRRSV 的复制。
因此，我们的研究结果初步揭示了猪TRIM32分子具有抗PRRSV 感染的作用，为进一步探究猪TRIM32抗HP-PRRSV 感染的机制奠定基础。
关键词：TRIM32；真核表达；抗病毒反应；HP-PRRSV 中图分类号：S852.4文献标志码：A文章编号：1674-6422（2023）03-0158-06Cloning, Expression and Functional Analysis of Porcine TRIM32LI Hui, ZHANG Yanbing, XIE Yixuan, LU Yan, XIANG Xiao, LIU Ke, WEI Jianchao, LI Zongjie,SHAO Donghua, LI Beibei, MA Zhiyong, QIU Yafeng(Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China)收稿日期：2020-02-19基金项目：中央级公益性科研院所基本科研业务费（2020JB04）；国家自然科学基金（31972693）；中国农业科学院青年英才项目（CAASQNYC-KYYJ-26）作者简介：李慧，女，硕士研究生，兽医学专业通信作者：邱亚峰，E-mail：****************.cn Abstract: TRIM32 is a member of TRIM family and plays an important role in host immune defense. However, it is little known about the role of porcine TRIM32 in innate antiviral response. In the present study, we fi rst cloned the porcine TRIM32 gene from pig lung by using RT-PCR, and the recombinant plasmid pFlag-porTRIM32 was constructed by inserting the porcine TRIM32 gene into p3xFlag-CMV-14 vector. Subsequently, effect of porcine TRIM32 on PRRSV infection was determined in Marc145 cell line by the gain-of-function analysis through over expression of pFlag-porTRIM32. Moreover, we further set up the loss-of function analysis through knockdown expression of endogenous TRIM32 in Marc145 cell line using RNA interference. Our results demonstrated that the recombinant plasmid pFlag-porTRIM32 was successfully constructed and anti-Flag antibody recognized recombinant protein Flag-porTRIM32 transiently expressed in 293T cell line in Western blotting. Overexpression of porcine TRIM32 in Marc145 cells inhibited PRRSV infection in comparison to infected-vector-group. In contrast, knock-down expression of endogenous TRIM32 in Marc145 cell line increased virus titters compared with those in infected-NC group. Thus, the results of this study not only revealed that porcine TRIM32 had a role in anti-PRRSV infection but also provided a basis for understanding insights into effect of TRIM32 on anti-PRRSV infection.Key words: TRIM32; eukaryotic expression; antiviral response; HP-PRRSV2023,31(3):158-163李 慧，张彦兵，解艺璇，陆 艳，相 笑，刘 珂，魏建超，李宗杰，邵东华，李蓓蓓，马志永，邱亚峰（中国农业科学院上海兽医研究所，上海200241）· 159 ·李 慧等：猪TRIM32分子的克隆、表达及其功能分析第31卷第3期TRIM32（The tripartite motif 32）作为TRIM家族中的成员之一，与TRIM家族其他成员的结构相似，其N端具有典型的RBBC结构，即一个RING结构域、两个B-BOX结构域、一个Cioned-coin卷曲螺旋结构域，与此相连的为1个间隔片段和6个NHL 重复序列[1-2]。
文章编号:100025404(2004)1321171204 论著猪皮肤成纤维细胞的培养与鉴定丁生财1,陈意生1,魏 泓2,刘 萍3 (第三军医大学:1西南医院病理科,2实验动物中心,重庆400038,3新桥医院妇产科,重庆400037) 提 要:目的 研究猪内源性逆转录病毒在体内和体外的感染性,建立理想的细胞模型,为猪一人间跨种移植的生物安全性评价奠定良好的基础。
方法 运用组织块培养法、冷胰蛋白酶方法及热胰蛋白酶方法培养猪皮肤成纤维细胞,比较3种方法的优点和缺点。
结果 建立了猪皮肤成纤维细胞系组织块培养法优于胰酶法。
结论 应用组织块培养法培养猪皮肤成纤维细胞优于胰酶法,经济实惠、操作简便、易于掌握;并为研究PERV在体内和体外的感染性提供了理想的细胞模型,为评估猪一人间跨种移植的生物安全性提供一种新的方法;对建立动物克隆生产的皮肤体外培养模式具有重要的参考价值。
关键词:成纤维细胞;猪内源性逆转录病毒;异种移植;生物安全性;感染;培养;皮肤;猪 中图法分类号:R322.99;R329.2 文献标识码:ACultivation and identification of porcine skin fibroblastsDI NG Sheng2cai1,CHE N2Y i2sheng1,WEI H ong2,LI U Ping3(1Research Institute of Pathology,S outhwest H ospital,2Department of Lab2 oratory Animal Science,3Department of Obstetrics and G ynecology,X inqiao H ospital,400037,Third Military Medical University,Chongqing 400038,China) Abstract:Objective s T o establish an ideal cellular m odel for the study of in fection of porcine endogenous ret2 rovirus in intro and in vivo and to lay the foundation for evaluating the biological safety of xenotransplantation.Meth2 ods P orcine skin fibroblasts were cultured by tissue pieces,cold trypsin,and heat trypsin,respectively.The merit and defect of these methods were analyzed.Re sults P orcine skin fibroblast line was established and the tissue piece method was superior to trypsin methods.Conclusion Culture of porcine skin fibroblasts with tissue pieces is superi2 or to trypsin method,with the characteristics of low cost and easy operation.The established porcine skin fibroblast cell line supplies an ideal cell m odel for the study of in fection of porcine endogenous retrovirus in vitro and in vivo, provides a new method for the evaluation of biological safety of xenotransplantation,and plays an im portant role in the skin culture m odel of production of cloned animals. K ey w ords:fibroblast cell;porcine endogenous retrovirus;xenotransplantion;biological safety;in fection;cul2 ture;skin;pig 猪器官、组织和细胞作为猪一人间跨种移植的供体源具有广阔的前景,但其基因组载有猪内源性逆转录病毒前病毒序列,可能导致猪内源性逆转录病毒(porcine endogenous retrovirus,PERV)种间传播发生的潜在危险。
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Supplementary information is available on Nature’s World-Wide Web site()or as paper copy from the London editorial office of Nature.AcknowledgementsWe thank the technical staff of RIKEN GSC,M.Horishima,H.Ishizaki,N.Ota and Y.Seki for sequencing;T.Yada for ORF prediction;A.Toyoda for technical support on the sequence library preparation;C.Kawagoe for computer system support;and T.D.Taylor for discussion.This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovation Biosciences(ProBRAIN) of the Bio-oriented Technology Research Advancement Institution,and Grants-in-Aid for Scientific Research from the Japanese Ministry of Education,Science,Sports and Culture.Correspondence and requests for materials should be addressed to Y.S.(e-mail:sakaki@ims.u-tokyo.ac.jp)or H.I.(e-mail:iskw@biol.s.u-tokyo.ac.jp).The complete sequence and the annotated data are available on our website(http://buchnera.gsc.riken.go.jp/).The sequence has been deposited with DDBJ under accession number AP000398,AP001070and AP001071for chromosome,the pTrp plasmid and the pLeu plasmid,respectively.................................................................. Cloned pigs produced by nuclear transfer from adult somatic cellsIrina A.Polejaeva*,Shu-Hung Chen*,Todd D.Vaught*,Raymond L.Page*, June Mullins*,Suyapa Ball*,Yifan Dai*,Jeremy Boone*,Shawn Walker*,David L.Ayares*,Alan Colman†&Keith H.S.Campbell†‡*PPL Therapeutics Incorporated,1700Kraft Drive,Blacksburg,Virginia24060, USA†PPL Therapeutics,Roslin,Midlothian EH259PP,UK .............................................................................................................................................. Since thefirst report of live mammals produced by nuclear transfer from a cultured differentiated cell population in1995 (ref.1),successful development has been obtained in sheep2,3, cattle4,mice5and goats6using a variety of somatic cell types as nuclear donors.The methodology used for embryo reconstruc-tion in each of these species is essentially similar:diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on,or after transfer.In sheep2and goat6pre-activated oocytes have also proved successful as cytoplast recipi-ents.The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term.In pigs,nuclear transfer has been significantly less success-ful;a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte7;how-ever,no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetalfibroblasts as nuclear donors8,9. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors.Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.To date,the efficiency of somatic cell nuclear transfer,when measured as development to term as a proportion of oocytes used, has been very low(1–2%)10.A variety of factors probably contribute to this inefficiency.These include laboratory to laboratory varia-tion,oocyte source and quality,methods of embryo culture(which are more advanced in some species(such as cows)than others(such as pigs)),donor cell type,possible loss of somatic imprinting in the nuclei of the reconstructed embryo,failure to reprogram the transplanted nucleus adequately,andfinally,the failure of artificial methods of activation to emulate reproducibly those crucial mem-brane-mediated events that accompany fertilization.In the pig,there is the additional difficulty that several(Ͼ4)good quality embryos are required to induce and maintain a pregnancy11. As fully developmentally competent embryos are rare in nuclear transfer procedures,there is every chance of squandering those good embryos unless very large numbers of reconstructed embryos are transferred back into recipients.Even if it were possible in the pig to select good quality blastocysts for transfer(after,for example,the use of a temporary recipient),most blastocysts formed from reconstructed embryos in other species are not competent to proceed to term10.The co-transfer of reconstructed embryos with ‘helper’,unmanipulated embryos,parthenotes or tetraploid embryos has been suggested as an aid to inducing and maintaining pregnancy.However,studies in mice after zygote pronuclear injec-tion have suggested that the manipulated embryos are‘compro-mised’and selected against12.An alternative to the use of‘helper’embryos is the hormonal treatment of recipient sows to maintain pregnancy with low embryo numbers13.We cannot currently address all of the methodological problems,and,to improve our chances of success in pig nuclear ‡Present address:University of Nottingham,School of Biosciences,Sutton Bonington,Leicestershire LE125RN,UK.transfer,we chose to focus on four areas:activation,choice of donor cell,embryo culture,and induction and maintenance of pregnancy.In all species,when using MII oocytes as recipients,the method of activation is crucial for subsequent development.In the pig, although current activation protocols stimulate pronuclear formation,cleavage,and development to the blastocyst stage, both the frequency of development and the quality of the embryos produced are low14.A system that involves the use of fertilized zygotes as cytoplast recipients would bypass the inefficiencies of artificial activation procedures and might promote more successful development.The technique of pronuclear exchange between zygotes showed that the manipulations involved were compatible with development15;however,when donor nuclei from later devel-opmental stages were transferred there was restricted development16.One explanation is that factors required for devel-opment,which are absent in the donor nuclei,are removed with the pronuclei.But if a pronucleus-like structure could be produced from the donor nucleus,this might prove a suitable nuclear donor for transfer.Such a system was described in mice by Kwon and Kono17,whofirst fused mitotically arrested blastomere nuclei with enucleated MII oocytes.The reconstructed oocytes were subse-quently activated in the presence of cytochalasin B,preventing polar body extrusion and resulting in the formation of two diploid pseudo-pronuclei.Each pseudo-pronucleus was then transferred into an enucleated,in vivo produced zygote,which was transferred into a surrogate recipient for development to term.Effectively,this latter procedure mimics pronuclear exchange and allows theMIIMIIoocyteZygotedonorGranulosa celldonorPronuclearzygoteCentrifugeCultureCellselectionFigure1Representation of the double nuclear transfer procedure for the production ofviable piglets using cultured adult somatic granulosa cells as nuclear donors.The outercircle in all the oocytes and embryos denotes the zona pellucida;the inner circle denotesthe cell membrane.Figure2A litter offive live piglets derived by nuclear transfer using cultured adultgranulosa cells as nuclear donors.A total of72reconstructed embryos were transferred tothe surrogate sow.Table1Development of porcine embryosDouble NT Single NTCell isolate Pool1GR5GR8GR12GR21Z GR18GR18GR1GR8GR18 ................................................................................................................................................................................................................................................................................................................................................................... Cell treatment CI CI CI CI CI SS(74)SS(51)CI CI SS(51) No.of oocytes245344269291311193216123109N/A No.of attemptedreconstructions day1(%)183(75)217(63)207(77)226(78)221(71)122(63)192(89)94(76)83(76)N/ANo.of fused embryosday1(%)124(68)153(70)186(90)97(43)163(74)90(74)162(84)87(93)61(73)N/A No.of day-1embryos withsingle pronucleus(%)87(70)88(57)120(64)62(64)69(42)23(26)102(63)–––No.of reconstructedembryos day2745710561552245*–––No.of fused embryosday2(%)72(97)56(98)100(95)53(87)54(98)22(100)44(98)–––No.of embryostransferred to recipient725610053542244856139†Pregnancy(no.of fetusesobserved)+ve(3)−ve−ve−ve−ve+ve(6)−ve−ve−ve−veNo.of live births(%)5(7)000000000 ................................................................................................................................................................................................................................................................................................................................................................... Development of porcine embryos reconstructed using a single or double nuclear transfer protocol with adult granulosa cells cultured to confluence(CI),or serum starved(SS)as nuclear donors.*Insuffici9ent zygotes to reconstruct day1embryos.†Due to insufficient zygote numbers39day-1recosntructed embryos were transferred to a single recipient.formation of afinal reconstructed one-cell embryo whose membrane has been activated during fertilization.The use of cultured cell populations for the production of animals by nuclear transfer is now well documented in a number of species. We have considerable experience in the production of sheep and cattle from primary cell populations and genetically modified primary cell populations.Analysis of these studies has shown considerable variation in development between individual cell populations and at present has provided no definitive method for the identification of cell populations that are suitable for nuclear transfer.Factors that are thought to influence the suitability of a particular cell population include the effects of oxidative damage associated with cellular metabolism,genome instabilities and chro-mosomal pathologies.All of these factors may be influenced by the method of isolation and culture,and the number of population doublings in culture.On consideration of these factors and our previous observations,we chose to use granulosa cells as nuclear donors.Granulosa cells are suitable nuclear donors in cattle18,and require the minimum of manipulations to establish in culture. Because of differences between cell populations,we initially decided to use a pool of cells isolated from a group of four donors.In later experiments,cell populations from individual animals were also examined.To minimize the culture period,early passage,never-frozen cells were used.For embryo reconstruction,we attempted to minimize the potential inefficiencies at each step of the nuclear transfer procedure and adopted an approach that(1)uses in vivo derived material,(2) seeks to avoid artificial activation,and(3)minimizes the period of in vitro culture of manipulated embryos.To do this we used a two-stage nuclear transfer procedure modified from Kwon and Kono17 (Fig.1).In thefirst stage,donor cells were fused to in vivo derived, enucleated MII oocytes obtained from superovulated crossbred gilts.The pseudo-pronucleus formed in thefirst nuclear transfer embryo was then subsequently transplanted into an in vivo pro-duced,enucleated zygote(second nuclear transfer embryo).The second nuclear transfer reconstructed embryo was transferred to the oviduct of a synchronized sow within2h of fusion.Because of the expected low developmental rate,we transferred up to100recon-structed embryos to a single recipient.Each recipient was treated with pregnant mare serum gonadotropin(PMSG)and human chorionic gonadotropin(hCG)to maintain pregnancy13in the event that fewer than four reconstructed embryos were viable at implantation.Coordination of the cell-cycle stages of the recipient cytoplasm and the donor nucleus are essential for maintaining correct ploidy and preventing DNA damage in nuclear transfer recon-structed embryos19.Various combinations of donor and recipient cell-cycle stages can prevent DNA damage and uncoordinated DNA replication,and result in formation of a pseudo-pronucleus.It has been suggested that the use of MII oocytes may improve‘re-programming’of the donor genetic material owing to the occur-rence of nuclear envelope breakdown and premature chromosome condensation,thus exposing the donor chromatin to maternally derived oocyte factors involved in early development.To take advantage of this here,we used MII oocytes as cytoplast recipients for thefirst nuclear transfer embryo reconstruction.To maintain ploidy in this situation,we chose diploid donor nuclei as nuclear donors.Previous studies have suggested that diploid cells arrested in the G0phase of the cell cycle may be benefiingflow cytometry, we examined the cell-cycle distribution of porcine granulosa cells under three different culture conditions:sub-confluent actively growing,100%confluent,and cells starved of serum for48hours (see Figure in Supplementary Information).After serum starvation, the population contained a large proportion(7.2%)of cells with a DNA content lower than that consistent with a diploid cell(termed sub-G1).In contrast,in the population synchronized by contact inhibition,90.3%of the cells had a diploid DNA content(G1/G0) and there were fewer sub-G1cells(1.6%).We analysed DNA synthesis in serum-starved and contact-inhibited cell populations by5-bromo-2Ј-deoxyuridine(BrdU)incorporation.These experi-ments revealed that45%of the contact inhibited cell population compared with0%of the serum-starved population incorporated BrdU.An analysis of BrdU incorporation after an additional24h of contact inhibition revealed that the fraction of BrdU-positive cells was reduced to5%.These observations suggest that the diploid cells in the contact-inhibited granulosa cell population used as nuclear donors for embryo reconstruction contained a mixture of cell-cycle-arrested diploid cells(G1/G0)and unarrested diploid cells(G1), which were able to undergo a further round of DNA synthesis.InTable2Microsatellite analysis of pigs and cell donorsLoci S0059S0070S0122S0226SW24SW72SW840SW936TNFB ................................................................................................................................................................................................................................................................................................................................................................... Samples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ecipient(54B)152275178192111102N/A103158 15229518219895102109161 Boar13426517818011510212997164 156273182180115112129109185 ................................................................................................................................................................................................................................................................................................................................................................... Microsatellite analysis was performed on genomic DNA from the four individual populations of granulosa cells(PGR1,PGR2,PGR3,PGR4),the piglets(NTP1–5),the surrogate sow(54B)and the boar responsible for inseminating the zygote donors.Primers corresponding to nine polymorphic loci were used.Two numbers are shown for each sample at each locus,which represent the PCR product size for each of the two alleles at that particular locus.contrast,when the serum-starved populations were used as nuclear donors most the diploid cells were cell-cycle arrested(G1/G0). Production of thefirst nuclear transfer embryos requires activa-tion of the in vivo derived oocytes.Activation experiments carried out in control oocytes showed that electrical stimulation applied between51.5and60h after hCG administration,promoted similar cleavage and development to blastocyst(see Table in Supplementary Information).For embryo reconstruction,MII oocytes were col-lected46–54h after hCG and thefirst nuclear tranfer embryo reconstruction was carried out between50and58h after hCG. Reconstructed embryos were cultured overnight in NCSU-23 medium22;we then checked them for the presence of a pronucleus and used them for the second nuclear transfer embryo reconstruc-tion.The development of single nuclear transfer and double nuclear transfer embryos reconstructed from contact-inhibited and serum-starved granulosa populations were compared(see Table1).In total, 185single nuclear transfer embryos were transferred to3recipient sows and401double nuclear transfer embryos to7recipients.Two recipients of the double nuclear transfer embryos became pregnant as determined by ultrasound visualization of fetuses at day35of gestation.One of these maintained the pregnancy to term,andfive piglets(Fig.2)were delivered by Caesarean section on day116of gestation.The average birth weight of the piglets was2.72lb(range 2.28–3.08lb);this is about25%lower than that observed in the same population of pigs under natural mating conditions(average litter size average10.9,average birth weight3.6lb,range3.3–3.9lb). The live piglets were produced from a pooled population of cells derived from four animals.We carried out microsatellite analysis of genomic DNA from the various samples(Table2).The comparison of the pattern of alleles in the piglets with that of the granulosa cell populations indicated that three of the nuclear transfer piglets (NTP1,NTP2and NTP3)were derived from the porcine granulosa (PGR)1/cell line,as there was100%identity at all nine microsatellite markers.The other2nuclear transfer piglets(NTP4and NTP5) showed perfect identity with the genotype of the PGR4cell population.Allfive of the nuclear transfer piglets were significantly different from the surrogate mother(54B).Some of the loci(S0059, S0070,S0122and TNFB)were not highly polymorphic indicating a degree of homogeneity or inbreeding within the population of pigs used in these studies(all of which come from the same commercial supplier).We think that the principal reasons for the success of this modified nuclear transfer procedure in pigs is its lack of reliance on current artificial activation protocols and in vitro culture techniques.Although elaborate,the double nuclear transfer does not add another major inefficiency(the second step fusion is very efficient).Direct transfer of a somatic nucleus to an enucleated zygote will not work because(in addition to reprogramming difficulties)of the loss of important factors sequestered within the removed pronuclei.The cell population used successfully as nuclear donors in these experiments were not quiesced by serum starvation. Cell-cycle analysis showed that most cells in control cultures had a diploid DNA content,and a high percentage were able to undergo a further round of DNA synthesis suggesting that most cells in the population were in the G1phase of the cell cycle and not arrested in G0.Allfive of the pigs,now three months old,are extremely healthy, in contrast to the(usual)50%postnatal loss of nuclear transfer animals10.It is tempting then to speculate that this modified method may have general utility in other species,even those where single nuclear transfer has been shown to work.The successful development of nuclear transfer in pigs opens the door for the application of gene-targeting technology,thus allowing for very precise genetic modifications,including gene knockouts.We have recently reported gene targeting in cultured ovine somatic cells and the successful development to term of offspring produced by nuclear transfer using these cells20.In pigs, a gene of great interest for the application of knockout technology is that for␣-1,3-galactosyl transferase(␣-1,3-GT)—the enzyme responsible for adding the xenogeneic sugar,galactose␣-1,3-galac-tose,to the surface of porcine cells.This gene is inactive in certain monkeys and humans,and their blood contains anti-gal antibodies, which trigger(in monkeys)early rejection of transplanted organs21. We have achieved targeted disruption of the␣-1,3-GT gene in primary porcine cells(unpublished data)and this will allow the production of␣-1,3-GT-deficient pigs,whose organs should show improved resistance to rejection.Overcoming antibody-mediated rejection is thefirst critical step in improving xenograft survival, towards the ultimate goal of providing an unlimited supply of compatible pig organs for human transplantation.ⅪMethodsModified NCSU-23mediumThe published NCSU-23medium22was modified for use as a phosphate-buffered benchtop medium without NaHCO3.Physiological pH phosphate buffer is made using a 3:1molar ratio of dibasic to monobasic phosphate anions.These changes induced alterations in the Na and K concentrations,which were corrected by adjusting the NaCl and KCl concentrations to maintain osmolarity and Na/K ratio(all chemicals purchased from Sigma unless otherwise noted).Superovulation of donor gilts for collection of oocytes and zygotes Crossbred gilts(280–320lbs)were synchronized by oral administration of18–20mg Regu-Mate(Altrenogest,Hoechst)mixed into the feed.Regu-Mate was fed for5–14d using a scheme dependent on the stage of the oestrous cycle.Estrumate(250g,Bayer) was administered intramuscularly(i.m.)on the last day of the Regu-Mate treatment. Superovulation was induced with a single i.m.injection of1,500IU of PMSG(Diosynth) 15–17h after the last Regu-Mate feeding.One thousand units of hCG(Intervet America) were administered i.m.82h after the PMSG injection.We collected oocytes46–54h after the hCG injection by reverseflush of the oviducts using pre-warmed Dulbecco’s phosphate buffered saline(PBS)containing bovine serum albumin(BSA;4g l−1).For the collection of zygotes,24–36h after the hCG injection the gilts were either artificially inseminated or bred naturally.Weflushed zygotes from the oviduct52–54h after the hCG injection using PBS containing BSA(4g l−1).Isolation and culture of porcine granulosa cellsFollicularfluid was aspirated from2–8-mm diameter follicles of superovulated crossbred gilts(Large White(1/2),Landrace(1/4),White Duroc(1/4)),7–8months old,280–320lb, 28–51h post hCG injection.Granulosa cells were collected by centrifugation at1,040g for 10min,re-suspended in DMEM(Gibco),containing10%fetal calf serum(FCS;Summit Biotech),0.1mM non-essential amino acids(NEAA Gibco),2ng ml−1basicfibroblast growth factor(bFGF)(Beckton Dickinson)and6l ml−1Gentamycin(Sigma).Cells were expanded for several days and then cryo-preserved.For nuclear transfer,we plated the granulosa cells at1–5×104cells per35mm dish in DMEM medium supplemented with NEAA(0.1mM),bFGF(2ng ml−1)and10%FCS, and cultured them to100%confluency at37ЊC.For experiments where serum starvation was evaluated,cells were starved of serum for48–72h in DMEM containing0.5%FCS.We collected cells by trypsinization and stored them in suspension in modified NCSU-23 phosphate medium at38.5ЊC for20–120min,before use as nuclear donors. Activation of oocytesActivation of control oocytes was achieved by application of two1.0kV cm−1DC electric pulses for60s each at an interval of5s in activation medium(0.3M D-sorbitol supplemented with0.1mM Mg SO4and0.05mM CaCl2in H20).Reconstruction offirst nuclear transfer embryoRecovered oocytes were washed in PBS containing4g l−1BSA at38ЊC,and transferred to calcium-free phosphate-buffered NCSU-23medium at38ЊC for transport to the laboratory.For enucleation,we incubated the oocytes in calcium-free phosphate-buffered NCSU-23medium containing5g ml−1cytochalasin B(Sigma)and7.5g ml−1Hoechst 33342(Sigma)at38ЊC for20min.A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an18-M glass pipette(Humagen, Charlottesville,Virginia).We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.A single granulosa cell was placed below the zona pellucida in contact with each enucleated oocyte.The couplet was transferred to a fusion chamber(model no.BT-453,BTX Inc.,San Diego)containing700l of0.3M mannitol,0.1mM MgS04and0.1mM CaC12in deionized water.Fusion and activation were induced by application of an AC pulse of5V for5s followed by two DC pulses of 1.5kVCm−1for60s using an ECM2001Electrocell Manipulator(BTX Inc.,San Diego). Couplets were then washed in bicarbonate buffered NCSU-23medium,and incubated in this medium for0.5–1h at38.6ЊC in a humidified atmosphere consisting of5%CO2in air.We checked couplets for fusion at×300magnification using an inverted microscope. Fused embryos were given a second activation stimulus of two successive DC pulses of 1.2kV cm−1for60s each,and cultured overnight in NCSU medium20.。
