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Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

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Journal of Animal Science and Biotechnology 2023年第1期14卷 98-113页

作者： Hao Wu Wendi Zhou Haijun Liu Xudai Cui Wenkui Ma Haixin Wu Guangdong Li Likai Wang Jinlong Zhang Xiaosheng Zhang Pengyun Ji Zhengxing Lian Guoshi Liu National Engineering Laboratory for Animal Breeding Key Laboratory of Animal Genetics and Breeding of the Ministry of AgriculturalBeijing Key Laboratory for Animal Genetic ImprovementCollege of Animal Science and TechnologyChina Agricultural UniversityBeijing 100193China. Sany Institute of China Agricultural University Sanya 572025China. Institute of Animal Husbandry and Veterinary Academy of Agricultural Sciences of TianjinTianjin 300192China. Qingdao Senmiao Industrial Co. Ltd.Qingdao 266101China.

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock ***,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic *** indicates the potential epigenetic variations between *** methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic *** this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using *** investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these ***:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly ***(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,*** annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG ***:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT *** results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the *** novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

关键词： Acetylserotonin-O-methyltransferase Dairy goat DNA methylation Gene editing somatic cell nuclear transfer

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Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer

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National Science Review 2019年第1期6卷 101-108页

作者： Zhen Liu Yijun Cai Zhaodi Liao Yuting Xu Yan Wang Zhanyang Wang Xiaoyu Jiang Yuzhuo Li Yong Lu Yanhong Nie Xiaotong Zhang Chunyang Li Xinyan Bian Mu-ming Poo Hung-Chun Chang Qiang Sun Institute of Neuroscience CAS Center for Excellence in Brain Science and Intelligence Technology CAS Key Laboratory of Primate Neurobiology State Key Laboratory of Neuroscience Chinese Academy of Sciences Shanghai Research Center for Brain Science and Brain-inspired Technology

Cloning of macaque monkeys by somatic cell nucleus transfer(SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey(Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing(see accompanying paper).Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood m RNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.

关键词： macaque monkey cloning somatic cell nuclear transfer non-human primate models CRISPR/Cas9 gene editing circadian rhythm disorders

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Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

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Journal of Genetics and Genomics 2008年第9期35卷 559-568页

作者： Jinghe Liu Xingwei Liang Jiaqiao Zhu Liang Wei Yi Hou Da-Yuan Chen Qing-Yuan Sun State Key Laboratory of Reproductive Biology Institute of Zoology Chinese Academy of Sciences Beijing 100101 China Graduate School of Chinese Academy of Sciences Beijing 100101 China

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.

关键词： DNA methylation DNA methyltransferase epigenetic reprogramming somatic cell nuclear transfer cow

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Handmade cloning:recent advances,potential and pitfalls

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Journal of Animal Science and Biotechnology 2015年第4期6卷 378-387页

作者： Geetika Verma JS Arora RS Sethi CS Mukhopadhyay Ramneek Verma School of Animal Biotechnology Post Graduate Institute of Veterinary Education and ResearchGuru Angad Dev Veterinary and Animal Sciences University

Handmade cloning （HMC） is the most awaited, simple and micromanipulator-ffee version of somatic cell nuclear transfer （SCNT）. The requirement of expensive micromanipulators and skilled expertise is eliminated in this technique, proving it as a major revolution in the field of embryology. During the past years, many modifications have been incorporated in this technique to boost its efficiency. This alternative approach to micromanipulator based traditional cloning O-C） works wonder in generating comparable or even higher birth rates in addition to declining costs drastically and enabling cryopreservation. This technique is not only applicable to intraspecies nuclear transfer but also to interspecies nuclear transfer （iSCNT） thus permitting conservation of endangered species. It also offers unique possibilities for automation of SCNT which aims at production of transgenic animals that can cure certain human diseases by producing therapeutics hence, providing a healthier future for the wellbeing of humans. The present review aims at highlighting certain aspects of HMC including recent advancements in procedure and factors involved in elevating its efficiency besides covering the potentials and pitfalls of this technique

关键词： Handmade cloning Micromanipulator somatic cell nuclear transfer Transgenic Zona free cloning

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Generation of B cell-Deficient Pigs by Highly Efficient CRISPR/Cas9-Mediated Gene Targeting

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Journal of Genetics and Genomics 2015年第8期42卷 437-444页

作者： Fengjiao Chen Ying Wang Yilin Yuan Wei Zhang Zijian Ren Yong Jin Xiaorui Liu Qiang Xiong Qin Chen Manling Zhang Xiaokang Li Lihua Zhao Ze Li Zhaoqiang Wu Yanfei Zhang Feifei Hu Juan Huang Rongfeng Li Yifan Dai State Key Laboratory of Reproductive Medicine and Jiangsu Key Laboratory of Xenotransplantation Nanjing Medical University

Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated （Cas） system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts （PFFs） enabled inducing gene knock out （KO） in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination （HR）. With the aid of somatic cell nuclear transfer （SCNT） technology, three piglets with the biallelic lgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the lgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.

关键词： CRISPR/Cas9 system lgM heavy chain Pig genome editing B cell-deficiency somatic cell nuclear transfer

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A brief review of recent advances in stem cell biology

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Neural Regeneration Research 2014年第7期9卷 684-687页

作者： Jinhui Chen Libing Zhou Su-yue Pan Stark Neuroscience Research Institute and Department of Neurological Surgery Indiana University School of Medicine Guangdong-Hongkong-Macao Institute of CNS Regeneration (GHMICR) Jinan University Department of Neurology Nanfang Hospital Southern Medical University

Stem cells have the remarkable potential to develop into many different cell types, essentially with- out limit to replenish other cells as long as the person or animal is still alive, offering immense hope of curing Alzheimer＇s disease, repairing damaged spinal cords, treating kidney, liver and lung diseases and making damaged hearts whole. Until recently, scientists primarily worked with two kinds of stem cells from animals and humans： embryonic stem cells and non-embryonic ＂somatic＂ or ＂adult＂ stem cells. Recent breakthrough make it possible to convert or ＂reprogram＂ specialized adult cells to assume a stem stem-like cells with different technologies. The review will briefly dis- cuss the recent progresses in this area.

关键词： stem cell adult stem cell embryonic stem cell somatic cell nuclear transfer induced plu-ripotent stern cell stimulus-triggered acquisition of pluripotency

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Telomere elongation in parthenogenetic stem cells

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Protein & cell 2014年第1期5卷 8-11页

作者： Yu Yin Na Liu Xiaoying Ye Renpeng Guo Jie Hao Fang Wang Lin Liu State Key Laboratory of Medicinal Chemical Biology Department of Cell Biology and GeneticsCollege of Life SciencesNankai UniversityTianjin 300071China

Dear Editor, Parthenogenetic embryonic stem （pES） cells, generated from oocytes by artificial activation without involvement of fertilization, show differentiation and pluripotency as evi- denced by their capacity to generate germline chimeras and all pES pups by tetraploid embryo complementation, indi- cating the ability of pES cells to form all cell types in the body （Chen et al., 2009; Liu et al., 2011）. Indeed, pES cells can repair injured muscle （Koh et al., 2009） and cardiomyocytes with reduced risk of tumorigenesis （Liu et al., 2013）,

关键词： Telomere Length somatic cell nuclear transfer Relative Telomere Length Telomere Elongation Longe Telomere

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

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Frontiers in Biology 2007年第1期2卷 80-84页

作者： JI Jingjuan GUO Tonghang TONG Xianhong LUO Lihua ZHOU Guixiang FU Yingyun LIU Yusheng Center for Reproductive Medicine Anhui Medical University Affiliated Provincial HospitalHefei 230001China

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research *** 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 *** our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus *** cumulus cell embryos showed significantly higher development rates than the other two(Psomatic cells of different chilling durations showed no significant ***,fluorescence in situ hybridization(FISH)was conducted to detect nuclear derivation of the *** result showed that the nuclei of the inter-species cloned embryo cells came from *** 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 ***,it is feasible to detect DNA genotype through FISH.

关键词： therapeutic cloning nuclear transfer somatic cell nuclear transfer inter-species cloned embryo

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The ecological adaptability of cloned sheep to free-grazing in the Tengger Desert of Inner Mongolia,China

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Frontiers of Agricultural Science and Engineering 2014年第3期1卷 191-200页

作者： Xinxin LI Huijuan WANG Guanghua SU Zhuying WEI Chunling BAI Wuni-MENGHE Yanhui HOU Changqing YU Shorgan BOU Guangpeng LI The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology Key Laboratory of Herbivore Reproductive Biotechnology and Breeding of Ministry of AgricultureInner Mongolia UniversityHohhot 010070China The Inner Mongolia Rangeland Ecology Institute Alashan 750306China

Since the birth of the first cloned sheep,somatic cell nuclear transfer technology has been successfully used to clone a variety of *** livestock have no apparent health risks,and the quality and safety of the cloned animal products are similar to non-cloned *** social behavior and environmental adaptability of postnatal cloned animals,especially when used for grassland farm production purposes,is *** the present study,the cloned Dorper sheep equipped with GPS location devices were free-grazed in a harsh natural environment similar to conditions commonly experienced by Mongolian *** main findings of this research were as follows.(1)Under free-grazing conditions,the cloned sheep showed excellent climatic and ecological *** extreme temperature conditions ranging from–30 to 40°C,the cloned sheep maintained acceptable body condition and behaved as other sheep.(2)The cloned sheep quickly adapted from a herd feeding strategy to the harsh environment and quickly exhibited a grazing regimen as other free-grazing sheep.(3)The cloned sheep exhibited free-grazing patterns and social behavior as other sheep.(4)The cloned sheep in the harsh environment thrived and produced healthy ***,the cloned Dorper sheep exhibited excellent ecological adaptation,which is an important consideration for breeding meat sheep by *** Dorper sheep readily adapted to the free-grazing conditions on the Mongolian plateau grassland,which attests to their ability to withstand harsh environmental conditions.

关键词： somatic cell nuclear transfer free-grazing synchronization Dorper sheep cloned animal ecology

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Factors affecting early embryonic development in cattle:relevance for bovine cloning

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Frontiers of Agricultural Science and Engineering 2019年第1期6卷 33-41页

作者： Yanna DANG Kun ZHANG Laboratory of Mammalian Molecular Embryology Institute of Dairy ScienceCollege of Animal SciencesZhejiang University

Female infertility represents a major challenge for improving the production ef?ciency in the dairy industry. Historically, fertility has declined whereas milk yield has increased tremendously due to intensive genetic selection. In vivo evidence reveals about 60% pregnancy loss takes place during the ?rst month following fertilization. Meanwhile, early embryo development is signi?cant for somatic cell nuclear transfer in cattle as a large proportion of cloned embryos fail to develop beyond periimplantation stage. Oocyte quality is of utmost importance for the early embryo to develop to term for both fertilized and cloned embryos. Epigenetic reprogramming is a key process occurring after fertilization and critical roles of epigenetic modi?ers during preimplantation development are now clear. Incomplete epigenetic reprogramming is believed to be a major limitation to cloning ef?*** of cloned embryos with epigenetic modifying drugs(e.g., Trichostatin A) could greatly improve cloning ef?ciency in both mice and cattle. Recently, the rapid progress in high-throughput sequencing technologies has enabled detailed deciphering of the molecular mechanisms underlying these events. The robust ef?ciency of genomic editing tools also presents an alternative approach to the functional annotation of genes critical to early development.

关键词： bovine cloning embryo development somatic cell nuclear transfer X-inactive specific transcript

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