体细胞核移植克隆西藏濒危樟木牛

doi:10.16431/j.cnki.1671-7236.2025.12.019

摘要/Abstract

摘要： 【目的】西藏特有牛种樟木牛濒临灭绝，本研究旨在采用体细胞核移植技术开展抢救性保护，为高海拔地区濒危物种保护提供新的途径。【方法】采用酶消化法建立樟木牛耳缘成纤维细胞，通过体细胞核移植技术制备重构胚胎，并移植至代孕母牛进行体内发育。采集流产个体组织进行亚硫酸氢盐测序，对父系表达基因10-ε肌聚糖(paternally expressed gene 10-sarcoglycan epsilon，PEG10-SGCE)、胰岛素样生长因子2-H19印迹母源表达转录本(insulin-like growth factor 2-H19 imprinted maternally expressed transcript，IGF2-H19)和鸟苷酸结合蛋白α亚基(guanine nucleotide-binding protein alpha subunit，GNAS)印迹域开展甲基化分析。【结果】本研究成功制备核型正常的樟木牛耳缘成纤维细胞，作为体细胞核移植供体细胞。移植32枚体细胞核移植囊胚至21头代孕母牛子宫，其中12头受孕成功，5头发育到期并成功诞下克隆牛。短串联重复序列分析发现，克隆牛的核DNA与供体牛成纤维细胞的核DNA完全匹配。对妊娠270 d发生流产的2头胎儿进行分析发现，PEG10-SGCE、IGF2-H19和GNAS印迹域在流产胎儿胎盘中呈现高甲基化，且GNAS印迹域在流产胎儿不同的组织中则呈现或低或高甲基化的异常状况。【结论】体细胞核移植技术能够应用于濒危樟木牛的抢救性保护。

关键词: 樟木牛; 濒危; 物种保护; 体细胞核移植; 甲基化

Abstract: 【Objective】 Zhangmu cattle is endemic to Tibet but on the brink of extinction.In this study,a rescue conservation was conducted using somatic cell nuclear transfer (SCNT) technology to provide a paradigm for the conservation of endangered species in high-altitude regions. 【Method】 Ear margin fibroblast from Zhangmu cattle were established by enzyme digestion method,reconstructed embryos were generated via SCNT technique and then transferred to surrogate cows for in vivo development.Bisulfite sequencing was performed on imprinting domains,including paternally expressed gene 10-sarcoglycan epsilon (PEG10-SGCE),insulin-like growth factor 2-H19 imprinted maternally expressed transcript (IGF2-H19) and guanine nucleotide-binding protein alpha subunit (GNAS),in tissues from aborted fetuses. 【Result】 SCNT embryos were produced using ear margin fibroblast of Zhangmu cattle with normal karyotype as donor cells.A total of 32 cloned blastocysts were transferred into the uteri of 21 recipient cows,among which 12 were successfully pregnant and 5 gave birth to cloned cattle.Short tandem repeat analysis showed that the nuclear DNA of cloned cattle was completely matched with that of donor fibroblast.The PEG1-SGCE,IGF2-H19 and GNAS imprinted domains were hypermethylated in the placenta of 2 aborted fetuses that occurred abortion at 270 days of gestation,and the GNAS imprinted domain was abnormally methylated at either a low or high manner in different tissues of the aborted fetuses. 【Conclusion】 SCNT technology could be applied to the rescue conservation of the endangered Zhangmu cattle.

Key words: Zhangmu cattle; endangered; species conservation; somatic cell nuclear transfer; methylation

中图分类号:

S813.3

齐浩南, 霍浩楠, 曹磊, 张元庆, 黄永业, 余大为. 体细胞核移植克隆西藏濒危樟木牛[J]. 中国畜牧兽医, 2025, 52(12): 5740-5748.

QI Haonan, HUO Haonan, CAO Lei, ZHANG Yuanqing, HUANG Yongye, YU Dawei. Cloning of the Endangered Zhangmu Cattle via Somatic Cell Nuclear Transfer in Tibet[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5740-5748.

参考文献

[1] 刘诗平.绝不让雪域高原濒危种质资源灭失——西藏阿沛甲咂牛与樟木牛抢救性保种全面展开[J].中国畜牧业，2022，24:10-11.
LIU S P.Never let the endangered germplasm resources be lost in the snowy plateau—Tibet Apeijiaza cattle and Zhangmu cattle rescue and conservation are fully launched[J].China Animal Industry，2022,24:10-11.(in Chinese)
[2] 刘鑫锋.全基因组重测序分析揭示中国3个濒危地方黄牛品种的适应性[D].杨凌：西北农林科技大学，2011.
LIU X F.Whole genome resequencing reveals adaptability of three endangered local cattle breeds in China[D].Yangling：Northwest A&F University，2011.(in Chinese)
[3] 王维佳,李道全,刘建雷,等.体细胞核移植技术及应用[J].黑龙江动物繁殖，2024，32(5):25-31.
WANG W J，LI D Q，LIU J L，et al.Somatic cell nuclear transfer technology and application[J].Heilongjiang Journal of Animal Reproduction，2024，32(5):25-31.(in Chinese)
[4] PABLO J R，JOSE B C.Bovine somatic cell nuclear transfer [J].Methods in Molecular Biology，2010，636:155-177.
[5] WILMUT I,BEAUJEAN N,DE SOUSA P A，et al.Somatic cell nuclear transfer[J].Nature，2002，419(6907):583-586.
[6] LI J，YU D，WANG J，et al.Identification of the porcine IG-DMR and abnormal imprinting of DLK1-DIO3 in cloned pigs[J].Frontiers in Cell and Developmental Biology，2022，10:964045.
[7] LI Y，SUN Q.Epigenetic manipulation to improve mouse SCNT embryonic development[J].Frontiers in Genetics，2022，13：932867.
[8] ZHANG Y，ZHANG C，CHEN W，et al.The landscape of allelic expression and DNA methylation at the bovine SGCE/PEG10 locus[J].Animal Genetics，2024，55(3)：452-456.
[9] LU X，YANG S，JIE M，et al.Folate deficiency disturbs PEG10 methylation modifications in human spina bifida [J].Pediatric Research，2022，92(4)：987-994.
[10] SANDOVICI I，FERNANDEZ-TWINN D S，CAMPBELL N，et al.Overexpression of IGF2-derived miR483 inhibits IGF1 expression and leads to developmental growth restriction and metabolic dysfunction in mice[J].Cell Reports，2024，43(9)：114750.
[11] LIAO J，SONG S，GUSSCOTT S，et al.Establishment of paternal methylation imprint at the H19/IGF2 imprinting control region[J]. Science Advances，2023，9(36)：eadi2050.
[12] YILDIZ I，SAGLIKER Y，DEMIRHAN O，et al.International evaluation of unrecognizably uglifying human faces in late and severe secondary hyperparathyroidism in chronic kidney disease[J].Journal of Renal Nutrition，2012，22(1)：157-161.
[13] YANG W，ZUO Y，ZHANG N，et al.GNAS locus:Bone related diseases and mouse models [J].Frontiers in Endocrinology，2023，14：1255864.
[14] 余大为.α干扰素在牛转基因细胞和核移植胚胎中的表达与功能研究[D].北京：中国农业科学院,2012.
YU D W.Studies on expression and function of interferon-α gene in the transgenic cells and nuclear transfer embroysof cattle[D].Beijing：Chinese Academy of Agricultural Sciences,2012.(in Chinese)
[15] COOMBER N,DAVID VA,O’BRIEN S J，et al.Validation of a short tandem repeat multiplex typing system for genetic individualization of domestic cat samples [J]. Croatian Medical Journal，2007，48(4)：547-555.
[16] SHIORI G，TERUAKI T，HIRONAGA K，et al.X monosomy in the endangered Kiso horse breed detected by a parentage test using sex chromosome linked genes and microsatellites [J]. The Journal of Veterinary Medical Science，2019，81(1)：91-94.
[17] 赵文轩,李俊雅,高晨,等,蒙山牛诱导多能干细胞的建立[J].畜牧兽医学报，2025,56(4):1731-1743.
ZHAO W X，LI J Y，GAO C，et al.Establishment and pluripotency analysis of induced pluripotent stem cells from Mengshan cattle[J].Acta Veterinaria et Zootechnica Sinica，2025,56(4):1731-1743.(in Chinese)
[18] 曹嘉程，薛丽娥，蓝群，等.表观遗传修饰影响哺乳动物体细胞核移植效率的研究进展[J].中国畜牧杂志，2023，59(3)：7-15.
CAO J C，XUE L E，LAN Q，et al.Research progress on the effect of epigenetic modification affecting the efficiency of mammalian somatic cell nuclear transfer [J].Chinese Journal of Animal Science，2023，59(3)：7-15.(in Chinese)
[19] ZHAO X，WU S，YUN Y，et al.Integrating transcriptomics,proteomics,and metabolomics to investigate the mechanism of fetal placental overgrowth in somatic cell nuclear transfer cattle[J].International Journal of Molecular Sciences，2024，25(17)：9388.
[20] PAIKER M，KHAN K，MISHRA D，et al.Morphological,morphometric,and histological evaluation of the placenta in cases of intrauterine fetal death[J].Cureus，2024，16(6)：e62871.
[21] LI Z K,WANG L B,WANG L Y,et al.Adult bi-paternal offspring generated through direct modification of imprinted genes in mammals[J].Cell Stem Cell，2025，32(3)：361-374.
[22] LU X,ZHANG Y,WANG L,et al.Evolutionary epigenomic analyses in mammalian early embryos reveal species-specific innovations and conserved principles of imprinting[J].Science Advances,2021，7(48):eabi6178.
[23] HALLIWELL D O，HONING F，BAGBY S，et al.Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing[J].Communications Biology，2025，8(1):243.