猪体细胞核移植效率的提高策略

doi:10.16431/j.cnki.1671-7236.2025.12.027

摘要/Abstract

摘要： 猪体细胞核移植(somatic cell nuclear transfer，SCNT)是将猪的体细胞核移植到去核卵母细胞中，通过激活重构胚胎使其发育为新个体的无性繁殖技术，在生产克隆猪及推动畜牧育种与生物医学研究方面起着重要作用，但猪体细胞核移植的供体细胞和受体卵母细胞选择与培养的复杂性、克隆效率低等原因导致其移植效率低下(＜5%)。作者系统综述了提高猪体细胞核移植效率的策略：①精准去核技术，如通过激光辅助、偏振光显微术等提高去核率；②供体细胞与卵母细胞优化，如低传代胎儿成纤维细胞、细胞周期同步化、动态激素组合培养等；③融合与激活方法创新，如电融合参数优化、锌离子调控等；④表观遗传挽救，如过表达重编程因子MBD3、抑制H3K9 me3修饰等。未来需要通过多技术协同(如代谢调控联合表观修饰)进一步突破发育效率瓶颈，推动猪克隆技术在畜牧生产与异种器官移植中的产业化应用。

关键词: 猪; 体细胞核移植; 供体细胞; 卵母细胞; 细胞周期; 能量代谢

Abstract: Somatic cell nuclear transfer (SCNT) in pigs involves transferring the nucleus of somatic cell from pigs into enucleated oocytes,activating the reconstructed embryo to develop into a new individual through asexual reproduction,it plays a significant role in producing cloned pigs and advancing livestock breeding and biomedical research.However,the complexity of selecting and culturing donor cells and recipient oocytes in SCNT of pigs,coupled with low cloning efficiency,results in the low transfer efficiency (less than 5%).The author systematically reviews the strategies to enhance the efficiency of SCNT in pigs:①Precise enucleation techniques,such as laser-assisted,polarized light microscopy to improve enucleation rate.②Optimization of donor cells and oocytes,including low-passage fetal fibroblasts,cell cycle synchronization,dynamic hormone combination culture,etc.③Innovation in fusion and activation methods,including the optimization of electrofusion parameters,zinc ion regulation,etc.④Epigenetic rescue,such as overexpression of reprogramming factor MBD3 and inhibition of H3K9 me3 modification.In the future,it is necessary to further overcome the bottleneck of developmental efficiency through multi-technology collaboration,such as metabolic regulation combined with epigenetic modification,and promote the industrial application of cloning technology of pigs in livestock production and xenotransplantation.

Key words: pigs; somatic cell nuclear transfer; donor cells; oocyte; cell cycle; energy metabolism

中图分类号:

S814.8

吴兴萍, 张海琳, 刘雪芹, 丁玉春, 丁川翔, 罗林, 吴梦. 猪体细胞核移植效率的提高策略[J]. 中国畜牧兽医, 2025, 52(12): 5829-5838.

WU Xingping, ZHANG Hailin, LIU Xueqin, DING Yuchun, DING Chuanxiang, LUO Lin, WU Meng. Strategies for Improving the Efficiency of Somatic Cell Nuclear Transfer in Pigs[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5829-5838.

参考文献

[1] SRIRATTANA K,KANEDA M,PARNPAI R.Strategies to improve the efficiency of somatic cell nuclear transfer[J].International Journal of Molecular Sciences,2022,23(4)：1969.
[2] VAJTA G.Cloning:A sleeping beauty awaiting the kiss?[J].Cell Reprogram,2018,20(3):145-156.
[3] GLANZNER W G,DE MACEDO M P,GUTIERREZ K,et al.Enhancement of chromatin and epigenetic reprogramming in porcine SCNT embryos-progresses and perspectives[J].Frontiers in Cell and Developmental Biology,2022,10:940197.
[4] POLEJAEVA I A,CHEN S H,VAUGHT T D,et al.Cloned pigs produced by nuclear transfer from adult somatic cells[J].Nature,2000,407(6800):86-90.
[5] LEE K,UH K,FARRELL K.Current progress of genome editing in livestock[J]. Theriogenology,2020,150:229-235.
[6] LOI P,IUSO D,CZERNIK M,et al.A new,dynamic era for somatic cell nuclear transfer?[J].Trends in Biotechnology,2016,34(10):791-797.
[7] DANG-NGUYEN T Q,WELLS D,HARAGUCHI S,et al.Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development[J].Journal of Reproduction and Development,2020,66(3):281-286.
[8] JANG G,JEON H Y,KO K H,et al.Developmental competence and gene expression in preimplantation bovine embryos derived from somatic cell nuclear transfer using different donor cells[J].Zygote,2005,13(3):187-195.
[9] TANG Y,ZHANG Y,LIU L,et al.Glycine and melatonin improve preimplantation development of porcine oocytes vitrified at the germinal vesicle stage[J].Frontiers in Cell and Developmental Biology,2022,10:856486.
[10] PARK M J,LEE S E,KIM E Y,et al.Effective oocyte vitrification and survival techniques for bovine somatic cell nuclear transfer[J].Cell Reprogram,2015,17(3):199-210.
[11] SPARMAN M L,TACHIBANA M,MITALIPOV S M.Cloning of non-human primates:The road "less traveled by"[J].International Journal of Developmental Biology,2010,54(11-12):1671-1678.
[12] MITALIPOV S M,YEOMAN R R,NUSSER K D,et al.Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells[J].Biologyof Reproduction,2002,66(5):1367-1373.
[13] ZHOU Q,YANG S H,DING C H,et al.A comparative approach to somatic cell nuclear transfer in the Rhesus monkey[J].Human Reproduction,2006,21(10):2564-2571.
[14] ZHAO Q,QIU J,FENG Z,et al.Robotic label-free precise oocyte enucleation for improving developmental competence of cloned embryos[J].IEEE Transactions on Biomedical Engineering,2021,68(8):2348-2359.
[15] LEE J E,LEE J Y,PARK C H,et al.Cryopreserved human oocytes and cord blood cells can produce somatic cell nuclear transfer-derived pluripotent stem cells with a homozygous Hla type[J].Stem Cell Reports,2020,15(1):171-184.
[16] IWAMOTO D,YAMAGATA K,KISHI M,et al.Early development of cloned bovine embryos produced from oocytes enucleated by fluorescence metaphase Ⅱ imaging using a conventional halogen-lamp microscope[J].Cell Reprogram,2015,17(2):106-114.
[17] BERNARDING J,BRUNS C,PREDIGER I,et al.Detection of sub-nmol amounts of the antiviral drug favipiravir in ¹⁹F MRI using photo-chemically induced dynamic nuclear polarization[J].Scientific Reports,2024,14(1):1527.
[18] MAENG G,GONG W,DAS S,et al.ETV2-null porcine embryos survive to post-implantation following incomplete enucleation[J].Reproduction,2020,159(5):539-547.
[19] SO C,MENELAOU K,URAJI J,et al.Mechanism of spindle pole organization and instability in human oocytes[J].Science,2022,375(6581):eabj3944.
[20] CAAMANO J N,MASIDE C,GIL M A,et al.Use of polarized light microscopy in porcine reproductive technologies[J].Theriogenology,2011,76(4):669-677.
[21] RISSI V B,GLANZNER W G,MUJICA L K,et al.Effect of cell cycle interactions and inhibition of histone deacetylases on development of porcine embryos produced by nuclear transfer[J].Cell Reprogram,2016,18(1):8-16.
[22] LEE S C,LEE W J,SON Y B,et al.Trichostatin A-induced epigenetic modifications and their influence on the development of porcine cloned embryos derived from bone marrow-mesenchymal stem cells[J].International Journal of Molecular Sciences,2025,26(5)：2359.
[23] ZHAI Y,LI W,ZHANG Z,et al.Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs[J].Molecular Reproduction and Development,2018,85(1):26-37.
[24] ZHANG Y T,YAO W,CHAI M J,et al.Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer[J].Journal of Veterinary Science,2022,23(2):e40.
[25] JIAO D,CHENG W,ZHANG X,et al.Improving porcine SCNT efficiency by selecting donor cells size[J].Cell Cycle,2021,20(21):2264-2277.
[26] CHO J,BHUIYAN M M,SHIN S,et al.Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell[J].Journal of Veterinary Medical Science,2004,66(12):1567-1573.
[27] WANG H,AO H,PAN Q,et al.Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos[J].Science China-Life Sciences,2007,50(2):178-185.
[28] KRAUSE M,GANSER C,KOBAYASHI E,et al.The Lewis GFP transgenic rat strain is a useful cell donor for neural transplantation[J].Cell Transplant,2012,21(9):1837-1851.
[29] MONIKA S,NARESH L,PRABHAT P,et al.An update:Reproductive handmade cloning of water buffalo (Bubalus bubalis)[J].Animal Reproduction Science,2018,197:1-9.
[30] CAROLINA G,CARLOS F,TEREZA C,et al.Production of bovine embryos and calves cloned by nuclear transfer using mesenchymal stem cells from amniotic fluid and adipose tissue[J].Cell Reprogram,2016,18(2):127-136.
[31] LIU H,PENG H,LIU F,et al.The expression of β-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos[J].In Vitro Cellular and Developmental Biology.Animal,2016,52(5):555-561.
[32] WILMUT I,SCHNIEKE A E,MCWHIR J,et al.Viable offspring derived from fetal and adult mammalian cells[J].Cloning Stem Cells,2007,9(1):3-7.
[33] SUBEDAR O D,CHIU L,WALDMAN S D.Cell cycle synchronization of primary articular chondrocytes enhances chondrogenesis[J].Cartilage,2021,12(4):526-535.
[34] GOISSIS M D,CAETANO H V,MARQUES M G,et al.Effects of serum deprivation and cycloheximide on cell cycle of low and high passage porcine fetal fibroblasts[J].Reproduction in Domestic Animals,2007,42(6):660-663.
[35] AKSHEY Y S,MALAKAR D,DE A K,et al.Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos[J].Theriogenology,2011,75(8):1516-1524.
[36] HYUN H,LEE S E,SON Y J,et al.Cell synchronization by rapamycin improves the developmental competence of porcine SCNT embryos[J].Cell Reprogram,2016,18(3):195-205.
[37] GILCHRIST R B,THOMPSON J G.Oocyte maturation:Emerging concepts and technologies to improve developmental potential in vitro[J].Theriogenology,2007,67(1):6-15.
[38] YUAN Y,SPATE L D,REDEL B K,et al.Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation[J].Proceedings of the National Academy of Sciences of the United States of America,2017,114(29):E5796-E5804.
[39] MAEDOMARI N,KIKUCHI K,OZAWA M,et al.Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte maturation affects fertilization and embryonic development in vitro[J].Theriogenology,2007,67(5):983-993.
[40] MARCHAL R,TOMANEK M,TERQUI M,et al.Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes[J].Molecular Reproduction and Development,2001,60(1):65-73.
[41] LEE Y,LEE H,PARK B,et al.Alpha-linolenic acid treatment during oocyte maturation enhances embryonic development by influencing mitogen-activated protein kinase activity and intraoocyte glutathione content in pigs[J].Journal of Animal Science,2016,94(8):3255-3263.
[42] LEE Y,LEE J,HYUN S H,et al.In vitro maturation using alpha-MEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer[J].Journal of Veterinary Science,2022,23(2):e31.
[43] CAI L,JEONG Y W,JIN Y X,et al.Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos[J].PLoS One,2020,15(3):e0230247.
[44] LEE Y,SHIM J,KO N,et al.Effect of alanine supplementation during in vitro maturation on oocyte maturation and embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs[J].Theriogenology,2019,127:80-87.
[45] RIDLO M R,KIM E H,TAWEECHAIPAISANKUL A,et al.Adiponectin improves in vitro development of cloned porcine embryos by reducing endoplasmic reticulum stress and apoptosis[J].Animals (Basel),2021,11(2)：473.
[46] HUANG L,KIM M Y,CHO J Y.Immunopharmacological activities of luteolin in chronic diseases[J].International Journal of Molecular Sciences,2023,24(3)：2136.
[47] JEONG P S,YANG H J,JEON S B,et al.Luteolin supplementation during porcine oocyte maturation improves the developmental competence of parthenogenetic activation and cloned embryos[J].PeerJ,2023,11:e15618.
[48] SADAT T S,FARASHAHI Y E,AGHARAHIMI A,et al.Cumulus cells conditioned medium facilitates germ cell differentiation from human embryonic stem cells:An experimental study[J].International Journal of Reproductive Biomedicine,2025,23(1):33-44.
[49] GLANZNER W G,RISSI V B,BORDIGNON V.Somatic cell nuclear transfer in pigs[J].Methods in Molecular Biology,2023,2647:197-210.
[50] WAKAYAMA T,PERRY A C,ZUCCOTTI M,et al.Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei[J].Nature,1998,394(6691):369-374.
[51] MCGRATH J,SOLTER D.Nuclear transplantation in the mouse embryo by microsurgery and cell fusion[J].Science,1983,220(4603):1300-1302.
[52] LEE E,SONG K.Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal[J].Journal of Veterinary Science,2007,8(4):415-421.
[53] SUTOVSKY P,MANANDHAR G,MCCAULEY T C,et al.Proteasomal interference prevents zona pellucida penetration and fertilization in mammals[J].Biology of Reproduction,2004,71(5):1625-1637.
[54] WHITWORTH K M,LI R,SPATE L D,et al.Method of oocyte activation affects cloning efficiency in pigs[J].Molecular Reproduction and Development,2009,76(5):490-500.
[55] ZHANG L,CHAO C H,JAEGER L A,et al.Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1[J].Biology of Reproduction,2018,98(4):510-519.
[56] MIYAZAKI S.Early and later studies on action potential and fertilization potential of echinoderm oocytes and Ca²⁺ response of mammalian oocytes[J].Methods in Cell Biology,2019,151:13-20.
[57] WILLIAMS C J.Signalling mechanisms of mammalian oocyte activation[J].Human Reproduction Update,2002,8(4):313-321.
[58] JONES K T.Turning it on and off:M-phase promoting factor during meiotic maturation and fertilization[J].Molecular Human Reproduction,2004,10(1):1-5.
[59] LEE K,DAVIS A,ZHANG L,et al.Pig oocyte activation using a Zn²⁺ chelator,TPEN[J].Theriogenology,2015,84(6):1024-1032.
[60] ZHAO M H,KIM N H,CUI X S.Zinc depletion activates porcine metaphase Ⅱ oocytes independently of the protein kinase C pathway[J].In Vitro Cellular & Developmental Biology-Animal,2014,50(10):945-951.
[61] DE MACEDO M P,GLANZNER W G,RISSI V B,et al.A fast and reliable protocol for activation of porcine oocytes[J].Theriogenology,2019,123:22-29.
[62] FERNANDES C B,DEVITO L G,MARTINS L R,et al.Artificial activation of bovine and equine oocytes with cycloheximide,roscovitine,strontium,or 6-dimethylaminopurine in low or high calcium concentrations[J].Zygote,2014,22(3):387-394.
[63] FERRER-BUITRAGO M,BONTE D,DE SUTTER P,et al.Single Ca²⁺ transients vs oscillatory Ca²⁺ signaling for assisted oocyte activation:Limitations and benefits[J].Reproduction,2018,155(2):R105-R119.
[64] SWANN K.The role of Ca²⁺ in oocyte activation during in vitro fertilization:Insights into potential therapies for rescuing failed fertilization[J].Biochimica et Biophysica Acta-Molecular Cell Research,2018,1865(11 PtB):1830-1837.
[65] FELMER R,ARIAS M E.Activation treatment of recipient oocytes affects the subsequent development and ploidy of bovine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos[J].Molecular Reproduction and Development,2015,82(6):441-449.
[66] BORGES A A,SANTOS M,NASCIMENTO L E,et al.Production of collared peccary (Pecari tajacu Linnaeus,1758) parthenogenic embryos following different oocyte chemical activation and in vitro maturation conditions[J].Theriogenology,2020,142:320-327.
[67] CHEN C,SUN T,YIN M,et al.Ionomycin-induced mouse oocyte activation can disrupt preimplantation embryo development through increased reactive oxygen species reaction and DNA damage[J].Molecular Human Reproduction,2020,26(10):773-783.
[68] WU G Q,JIA B Y,LI J J,et al.L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs[J].Theriogenology,2011,76(5):785-793.
[69] WU H,LEVALLEY P J,LUO T,et al.Manipulation of glutathione-mediated degradation of thiol-maleimide conjugates[J].Bioconjugate Chemistry,2018,29(11):3595-3605.
[70] PARK S H,JEONG P S,JOO Y E,et al.Luteolin orchestrates porcine oocyte meiotic progression by maintaining organelle dynamics under oxidative stress[J].Frontiers in Cell and Developmental Biology,2021,9:689826.
[71] WANG L,TANG J,WANG L,et al.Oxidative stress in oocyte aging and female reproduction[J].Journal of Cellular Physiology,2021,236(12):7966-7983.
[72] NIKOLOFF N,CAMPAGNA A,LUCHETTI C,et al.Effects of EPA on bovine oocytes matured in vitro with antioxidants:Impact on the lipid content of oocytes and early embryo development[J].Theriogenology,2020,146:152-161.
[73] ZHAN C,CAO X,ZHANG T,et al.Melatonin protects porcine oocyte from copper exposure potentially by reducing oxidative stress potentially through the Nrf2 pathway[J].Theriogenology,2022,193:1-10.
[74] LEE S,PARK E J,MOON J H,et al.Sequential treatment with resveratrol-trolox improves development of porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer[J].Theriogenology,2015,84(1):145-154.
[75] JEON Y,YOON J D,CAI L,et al.Supplementation of zinc on oocyte in vitro maturation improves preimplatation embryonic development in pigs[J].Theriogenology,2014,82(6):866-874.
[76] KUMBHA R,HOSNY N,MATSON A,et al.Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method[J].Research in Veterinary Science,2020,128:59-68.
[77] JIN J X,LEE S,KHOIRINAYA C,et al.Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer[J].Journal of Animal Science,2016,94(3):963-970.
[78] LUO C,WANG Z,WANG J,et al.Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism[J].Science China-Life Sciences,2022,65(10):2076-2092.
[79] CECIL R F,CHEN P R,BENNE J A,et al.Chemical simulation of hypoxia in donor cells improves development of somatic cell nuclear transfer-derived embryos and increases abundance of transcripts related to glycolysis[J].Molecular Reproduction and Development,2020,87(7):763-772.
[80] CAO J,DONG Y,LI Z,et al.Treatment of donor cells with oxidative phosphorylation inhibitor cpi enhances porcine cloned embryo development[J].Animals (Basel),2024,14(9)：1362.
[81] MAURICI N,PHAN T M,HENTY-RIDILLA J L,et al.Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation[J].Biology Preprint Archive,2024，2024：591564.
[82] WANG X,SHI J,CAI G,et al.Overexpression of MBD3 improves reprogramming of cloned pig embryos[J].Cell Reprogram,2019,21(5):221-228.
[83] HUANG R,SUI L,FU C,et al.HDAC11 inhibition disrupts porcine oocyte meiosis via regulating alpha-tubulin acetylation and histone modifications[J].Aging (Albany NY),2021,13(6):8849-8864.
[84] CAO L,DAI X,HUANG S,et al.Inhibition of Suv39 h1/2 expression improves the early development of Debao porcine somatic cell nuclear transfer embryos[J].Reproduction in Domestic Animals,2021,56(7):992-1003.
[85] KIM S J,KWON H S,KWON D K,et al.Production of transgenic porcine embryos reconstructed with induced pluripotent stem-like cells derived from porcine endogenous factors using piggybac system[J].Cell Reprogram,2019,21(1):26-36.
[86] KEEFER C L.Artificial cloning of domestic animals[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(29):8874-8878.
[87] EUN K,HWANG S U,KIM M,et al.Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer[J].Journal of Biotechnology,2022,17(7):e2100434.
[88] TELUGU B P,PARK K E,PARK C H.Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications[J].Mammalian Genome,2017,28(7-8):338-347.