Effect of Donor Cell on the Efficiency of Ovine Somatic Cell Cloning

doi:10.6048/j.issn.1001-4330.2020.01.022

Xinjiang Agricultural Sciences ›› 2020, Vol. 57 ›› Issue (1): 190-196.DOI: 10.6048/j.issn.1001-4330.2020.01.022

Effect of Donor Cell on the Efficiency of Ovine Somatic Cell Cloning

GUO Yanhua¹, XU Fangye¹, LI Yingli², ZHANG Yiyuan¹, WANG Limin¹, TANG Hong¹, PI Wenhui¹, ZHOU Ping¹,

1. State Key Laboratory of Sheep Genetic Improvement and Healthy Production/ Institute of Animal Husbandry and Veterinary, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi Xinjiang 832000, China;
2.Shihezi Veterinary Health and Quarantine Institute, Shihezi Xinjiang 832000, China

Received:2019-07-25 Online:2020-01-20 Published:2020-02-06
Correspondence author: ZHOU Ping(1974-),male, native of Leshan, doctor, professor, mainly engaged in research with somatic cell cloning and embryonic development,(E-mail)zhqxqf@163.com
Supported by:
Supported by the National Major Special Project on New Cultivation for Transgenic Organisms (2016zx08008-001);the National Natural Science Foundation of China (3166090049);Open Ssubject of Key Laboratory of Ovine Genetic Improvement and Healthy Production Laboratory (2013KLS05); the Ear-marked Fund of Academician in Production and Construction of Xinjiang (2014BB023,2016AG022,2016AG021)

供核细胞对绵羊体细胞克隆效率的影响

郭延华¹, 徐方野¹, 李迎利², 张译元¹, 王立民¹, 唐红¹, 皮文辉¹, 周平¹

1. 省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所,新疆石河子 832000;
2. 石河子市兽医卫生检疫所,新疆石河子 832000

通讯作者: 周平(1974-),男,四川乐山人,研究员,博士,研究方向为绵羊体细胞克隆与胚胎发育,(E-mail)zhpxqf@163.com
作者简介:郭延华(1983-),男,新疆喀什人,助理研究员,硕士,研究方向为绵羊体细胞克隆与表观遗传学,(E-mail)282437680@qq.com
基金资助:
国家转基因生物新品种培育重大专项(2016zx08008-001);国家自然科学基金(3166090049);绵羊繁育重点实验室开放课题 (2013KLS05);新疆生产建设兵团院士基金专项(2014BB023,2016AG022,2016AG021)

Abstract

Abstract: 【Objective】 In the production of cloned embryos, nuclear donor cells directly restrict the fusion number and developmental potential of focused embryos. Therefore, the purpose of this study is to investigate the static time, passage times and cycle treatment of donor cells, different cloned lines of donor cells and different sheep cells, and to determine the effect of single factor donor cells on cloned embryos by using reconstructed embryo fusion rate and blastocyst development rate as detection indexes.【Result】 The fusion rates of pelleted cells on 15-30 minutes in operation liquid drop was higher than longtime (about 2 h) pelleted cells after being removed from plates and re-suspension in centrifugal tube (90.10% vs78.84% )(P<0.01). Embryonic development competence had no significant difference (15.58% vs 14.65%)(P>0.05).Thawed fibroblast cells were cultured one to three passages and to full confluency on the plateau phase of cell growth curve 24 h and 48h, fusion rate of cytoplast-donor cell couplets had no significant difference (70.37%、76.03%、71.92% vs 72.97%)(P>0.05),but embryonic development competence rose gradually(5.96%, 9.06%, 15.85% vs 20.16%); Fusion rates and blastocyst rates of cloned embryos had significant difference(P＜0.05) or remarkable difference (P<0.01). On the different lines from mono adult fibroblast cell; coincidentally, it was same to the different lines from multiple adult fibroblast cell.【Conclusion】 These data showed that static time of resuspension somatic cells among 15-30 minutes in liquid drop, passage several times and prolong the G1 phase of somatic cell formed a confluent monolayer, and multiple somatic cells were conducive to the production of cloned embryos in ovine.

Key words: donor cell; static time; passage; cell line; cloned embryo

摘要： 【目的】在制作克隆胚胎时,供核细胞直接制约着重构胚的融合数量和发育潜力。研究供核细胞对绵羊体细胞对克隆效率的影响。【方法】从供核细胞的静置时间,研究传代次数与周期处理,供核细胞不同克隆株,不同羊源细胞等,以重构胚融合率和囊胚发育率为检测指标,判定单因素供核细胞对克隆胚胎的影响。【结果】在供核细胞静置时间方面,供核细胞静置15~30 min内完成重构胚制作的融合率极显著高于静置2 h左右的供核细胞组(90.10% vs 78.84%)(P< 0. 01),但囊胚率差异不显著(15.58%vs 14.65%)(P＞0.05);在供核细胞传代次数(d1~d3)和汇合期生长时间方面(24 and 48 h ),各组的融合率差异不显著(70.37%、76.03%、71.92% vs 72.97%)(P＞0.05),但囊胚率有增高的趋势(5.96%、9.06%、15.85% vs 20.16%);在同一供核细胞不同克隆株方面,供核细胞对融合率和囊胚率存在显著(P<0.05)或极显著(P<0.01)的影响;在不同个体方面,供核细胞株对融合率和囊胚率存在极显著的影响(P<0.01)。【结论】15~30 min内的静置供核细胞、传代3次并延长汇合期细胞生长时间(48 h),多选细胞株更有利于克隆胚的制作。

关键词: 供核细胞, 静置时间, 传代次数, 细胞株, 克隆胚胎

CLC Number:

S826
S816

GUO Yanhua, XU Fangye, LI Yingli, ZHANG Yiyuan, WANG Limin, TANG Hong, PI Wenhui, ZHOU Ping,. Effect of Donor Cell on the Efficiency of Ovine Somatic Cell Cloning[J]. Xinjiang Agricultural Sciences, 2020, 57(1): 190-196.

郭延华, 徐方野, 李迎利, 张译元, 王立民, 唐红, 皮文辉, 周平. 供核细胞对绵羊体细胞克隆效率的影响[J]. 新疆农业科学, 2020, 57(1): 190-196.

References

[1]Wilmut I, Schniek A E, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells [J]. Nature (London), 1997, 385(6619):810-813.
[2] Kues W A, Niemann H. Advances in farm animal transgenesis [J]. Preventive Veterinary Medicine, 2011, 102(2):146-156.
[3] Liu, Zhen, Cai, Yijun, Wang, Yan. Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer [J]. Cell, 2018,172:881-887
[4] Inoue K , Noda S , Ogonuki N , et al. Differential Developmental Ability of Embryos Cloned from Tissue-Specific Stem Cells[J]. Stem Cells (Miamisburg), 2007, 25(5):1279-1285.
[5] Wakayama S, Ohta H , Hikichi T, et al. Production of healthy cloned mice from bodies frozen at -20℃ for 16 years[J]. Proceedings of the National Academy of Sciences, 2008, 105(45):17318-17322.
[6] Bordignon V, Albornoz M , Colato C, et al. 30 Scriptaid treatment improves post-implantation development of sheep cloned embryos[J]. Reproduction, Fertility and Development, 2010, 23 (1):121-121.
[7] Vikram A, Bell G W, Jin-Wu N, et al. Predicting effective microRNA target sites in mammalian mRNAs [J]. eLife, 2015, (4):1-38
[8]郭延华,王立民,唐红,等.绵羊体细胞核移植融合效率影响因素的研究[J].新疆农业科学, 2014,51(1):136-142.
GUO Y H, WANG L M, TANG H, et al.The effect of repector, donor and pulsed strength on the fusion rate of somatic cell nuclear transfer in ovine [J]. Xinjiang Agricultural Sciences, 2014,51(1):136-142.
[9]郭延华,张译元,王立民,等.绵羊体细胞核移植去核前程序的优化[J].生物工程学报, 2017, 33(5): 730-742.
GUO Y H, ZHANG YY, WANG L M, et al. Program optimization before enucleation on ovine somatic cell nuclear transfer[J]. Chin J Biotech, 2017, 33(5): 730-742.
[10] Loi P , Ptak G , Barboni B , et al. Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells[J]. Nature Biotechnology, 2001, 19(10):962-964.
[11] Hosseini S M , Moulavi F , Foruzanfar M , et al. Effect of donor cell type and gender on the efficiency of in vitro sheep somatic cell cloning[J]. Small Ruminant Research, 2008, 78(1-3):162-168.
[12] Beaujean, N. Effect of Limited DNA Methylation Reprogramming in the Normal Sheep Embryo on Somatic Cell Nuclear Transfer [J]. Biology of Reproduction, 2004, 71(1):185-193.
[13]李甫强,周鸿鹰,羊惠君,等.差速贴壁法纯化分离GFP转基因小鼠骨髓间充质干细胞[J].四川大学学报(医学版),2006,37(2):301 - 304.
LI F Q, ZHOU H Y,YANG H J, et al. Isolation and purification of BMSCs of GFP Transgenic mouse using the method of adhering to culture plastic in different time [J].J Sichuan Univ (Med Sci Ed.), 2006, 37(2):301-304.
[14] Zakhartchenko V , Durcova-Hills G , Stojkovic M , et al. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts[J]. Reproduction, 1999, 115(2):325-331.
[15] Boquest, A. C. Flow Cytometric Cell Cycle Analysis of Cultured Porcine Fetal Fibroblast Cells[J]. Biology of Reproduction, 1999, 60(4):1013-1019.
[16] Sangho R, Hosup S, Woosuk H, et al. In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts.[J]. Reproduction Fertility & Development, 2000, 12(1-2):1-6.
[17] Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultureddonor cells [J]. Theriogenology, 62(3-4):512-521.
[18] Miranda, Moysés dos Santos, Bressan F F , Zecchin K G, et al. Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle[J]. Cloning and Stem Cells, 2009, 11(4):565-573.
[19] Libing M, Xiyu L , Fengmei W, et al. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos[J]. PLOS ONE, 2015, 10(8): 1-13.
[20] Kubota C, Yamakuchi H, Todoroki J, et al. Six cloned calves produced from adult fibroblast cells after long-term culture[J]. Proceedings of the National Academy of Sciences, 2000, 97(3):990-995.
[21] Loi, P. Nuclei of Nonviable Ovine Somatic Cells Develop into Lambs after Nuclear Transplantation [J]. Biology of Reproduction, 2002, 67(1):126-132.
[22] Iguma L T, Lisauskas S F C, Melo E O, et al. Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells [J]. Genetics and molecular research: GMR, 2005, 4(1):55-66.
[23] Wani N A , Hong S B . Source, treatment and type of nuclear donor cells influences in vitro and in vivo development of embryos cloned by somatic cell nuclear transfer in camel (Camelus dromedarius) [J]. Theriogenology,106(15):186-191.
[24] Zakhartchenko V, Mueller S, Alberio R, et al. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts [J]. Molecular Reproduction & Development, 2001, 60(3):362-369.

Effect of Donor Cell on the Efficiency of Ovine Somatic Cell Cloning

供核细胞对绵羊体细胞克隆效率的影响

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