绵羊iPS细胞诱导及转录组学分析

doi:10.6048/j.issn.1001-4330.2018.11.022

新疆农业科学 ›› 2018, Vol. 55 ›› Issue (11): 2142-2149.DOI: 10.6048/j.issn.1001-4330.2018.11.022

绵羊iPS细胞诱导及转录组学分析

张译元¹, 郭延华¹, 王聪慧¹, 唐红¹, 南海艳², 王立民¹, 周平¹

1.新疆农垦科学院畜牧兽医研究所/省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子 832000;
2.陕西西安市阎良区动物疾病预防控制中心,西安 710000

收稿日期:2018-09-12 发布日期:2019-02-22
通信作者: 王立民(1980-),男,内蒙古包头人,研究员,博士,研究方向为绵羊体细胞克隆与胚胎发育,E-mail:wanglm1980@126.com
作者简介:张译元(1988-),女,山东单县人,助理研究员,硕士,研究方向为转基因绵羊,E-mail:zhangyiyuan1988@sina.com;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
基金资助:
国家转基因重大专项“高产超细毛转基因羊新品种培育”(2016ZX08008001);兵团基础研究项目(2015AG018);兵团基础研究项目(2015AG011);新疆农垦科学院青年科学基金(YQJ201502)

Induction and Transcriptomics Analysis of Ovine iPS Cells

ZHANG Yi-yuan¹, GUO Yang-hua¹, WANG Cong-hui¹, TANG Hong¹, NAN Hai-yan², WANG Li-min¹, ZHOU Pin¹

Institute of Animal Husbandry and Veterinary Science, Xinjiang Academy of Agricultural And Land Reclamation Sciences / State Key Laboratory for Ovine Genetic Improvement and Healthy Breeding Technology of Province-Ministry Co-Construction, Shihezi Xinjiang 832000, China

Received:2018-09-12 Published:2019-02-22
Correspondence author: WANG Li-min(1980-）,male, Native of Baotou, professor, doctor, mainly engaged in research with somatic cell cloning and embryonic development,E-mail:wanglm1980@126.com
About author:ZHOU Ping(1974-),male, Native of Leshan, professor, doctor, mainly engaged in research with somatic cell cloning and embryonic development, E-mail:zhpxqf@163.com
Supported by:
Selection of High-yield and Fine-quality Peanuts Varieties and Study on the Cultivation(2016B01008-2)

摘要/Abstract

摘要： 【目的】建立绵羊(Ovis aries)诱导性多能干细胞系,为绵羊转基因育种、应用绵羊诱导性多能干细胞奠定基础。【方法】利用电转将OCT4、SOX2、KLF4、l-Myc、Lin28转染至绵羊成纤维细胞中并诱导培养至有光滑隆起、边缘整齐的细胞克隆,形态学、碱性磷酸酶(AP)染色、免疫荧光染色以及实时荧光定量PCR检测克隆细胞的生物学特性;利用高通量测序技术对诱导0、10、20、30 d的阳性细胞的差异表达基因进行筛选。【结果】克隆细胞中SOX2、OCT4、SSEA-1蛋白免疫荧光染色均呈阳性; OCT4、SOX2、Nanog基因表达量均极显著高于成纤维细胞;0和30 d转录组测序表明,被注释到GO数据库的差异表达基因有9 465个,其中,上调基因6 987个,下调基因2 478个;KEGG分析共有5 773个基因注释到259个通路中,其中,P53信号通路显著富集,且其在干细胞增殖方面起着非常重要的作用。【结论】初步成功建立了绵羊诱导性多能干细胞。

关键词: 绵羊; 诱导性多能干细胞(iPSCs); 重编程; 转录组测序

Abstract: 【Objective】 The aim of this study is to establish sheep (ovis aries) induced multifunctional cell lines in the hope of laying the foundation for sheep transgenic breeding.【Method】Colonies derived from ovine fibroblasts transfected with OCT4, SOX2, KLF4, 1-Myc and Lin 28 by electroporation displayed smooth domes with sharp edges when grown in human iPSCs medium. Morphology, alkaline phosphatase(AP) staining, immunofluorescence staining and real-time fluorescence quantitative PCR were used to detect the biological characteristics of cloned cells. Also, the experiment adopted the high fluxtranscriptome sequencing technology to screen the differentially expressed genes of 0 d, 10 d, 20 d and 30 d cloned cells.【Result】The results showed that the SOX2, OCT4, SSEA-1 protein and immunofluorescence staining of the cloned cells were positive. The expression of OCT4, SOX2, and Nanog genes in cloned cells were significantly higher than those in fibroblasts. The results of 0 d and 30 d transcriptom sequencing showed that 9,465 differentially expressed genes were annotated to the GO database, of which 6,987 were up-regulated and 2,478 were down-regulated. The KEGG analysis had a total of 5,773 genes annotated to 259 pathways, of which the P53 signaling pathway was significantly enriched. What happened here played a very important role in stem cell proliferation.【Conclusion】The above results suggest that ovine induced pluripotent stem cells have been successfully established, which had laid the foundation for further study and application of ovine induced pluripotent stem cells.

Key words: ovis aries; induced pluripotent stem cells (iPSCs); reprogramming; transcriptome sequencing

中图分类号:

S826.8

张译元, 郭延华, 王聪慧, 唐红, 南海艳, 王立民, 周平. 绵羊iPS细胞诱导及转录组学分析[J]. 新疆农业科学, 2018, 55(11): 2142-2149.

ZHANG Yi-yuan, GUO Yang-hua, WANG Cong-hui, TANG Hong, NAN Hai-yan, WANG Li-min, ZHOU Pin. Induction and Transcriptomics Analysis of Ovine iPS Cells[J]. Xinjiang Agricultural Sciences, 2018, 55(11): 2142-2149.

参考文献

[1] Molofsky, A. V. , Pardal, R. , & Morrison, S. J. . (2004). Diverse mechanisms regulate stem cell self-renewal. Current Opinion in Cell Biology, 16(6): 700-707.
[2] Odorico, J. S. , Kaufman, D. S. , & Thomson, J. A. (2001). Multilineage differentiation from human embryonic stem cell lines. Stem Cells, 19(3): 193-204.
[3] Takahashi, K., Tanabe, K., Ohnuki, M., Ichisaka, T., Tomoda, K. & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5): 861-872,
[4] Yu, J. , Vodyanik, M. A. , Smuga-Otto, K. , Antosiewicz-Bourget, J. , Frane, J. L. , & Tian, S. , et al. (2008). Induced pluripotent stem cell lines derived from human somatic cells. Obstetrical & Gynecological Survey, 63(3): 154-155.
[5] Zhao, X. Y. , Li, W. , Lv, Z. , Liu, L. , Tong, M. , & Hai, T. , et al. (2010). Ips cells produce viable mice through tetraploid complementation. China Basic Science,461(7260): 86-90.
[6] Liao, J. , Cui, C. , Chen, S. , Ren, J. , Chen, J. , & Gao, Y. , et al. (2009). Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell, 4(1):11-15.
[7] Liu H; Zhu F; Yong J; Zhang P; Hou P; Li H; Jiang W; Cai J; Liu M; Cui K; Qu X; Xiang T; Lu D; Chi X; Gao G; Ji W; Ding M; Deng H. (2008). Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell, 3(6): 587-590.
[8] Wu, Z. , Chen, J. , Ren, J. , Bao, L. , Liao, J. , & Cui, C. , et al. (2009). Generation of pig induced pluripotent stem cells with a drug-inducible system. Journal of Molecular Cell Biology, 1(1): 46-54.
[9] Bao, L. , He, L. , Chen, J. , Wu, Z. , Liao, J. , & Rao, L. , et al. (2011). Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Research, 21(4): 600-608.
[10] Takahashi, K., Okita, K., Nakagawa, M., & Yamanaka, S. (2006). Induction of pluripotent stem cells from fibroblast cultures. Nature Protocols, 51(15): 3,081-3,089.
[11] Liu, J. , Balehosur, D. , Murray, B. , Kelly, J. M. , Sumer, H. , & Verma, P. J. . (2012). Generation and characterization of reprogrammed sheep induced pluripotent stem cells. Theriogenology, 77(2): 338-346.
[12] Sartori, C. , Didomenico, A. I. , Thomson, A. J. , Milne, E. , & Whitelaw, C. B. A. . (2012). Ovine-induced pluripotent stem cells can contribute to chimeric lambs. Cellular Reprogramming, 14(1): 8-19.
[13]王峰,刘灿,潘传英.大家畜诱导多潜能干细胞(iPSCs)研究进展[J]. 农业生物技术学报,2014, 22(10): 1 286-1 297.
WANG Feng, LIU Can, PAN Chuang-ying. (2014). Progress of induced pluripotent stem cells (iPSCs) of big domestic animals [J]. Journal of Agricultural Biotechnology, 22(10):1,286-1,297. (in Chinese)
[14] Li, Y. , Cang, M. , Lee, A. S. , Zhang, K. , & Liu, D. . (2011). Reprogramming of sheep fibroblasts into pluripotency under a drug-inducible expression of mouse-derived defined factors. PLOS ONE, 6(1): e15947:1-8.
[15]陈倩,史庆华. iPS细胞的遗传安全性[J]. 遗传,2012, 34(3): 260-268.
CHEN Qian, SHI Qing-hua. (2012). The genetic safety of induced pluripotent stem (ips) cells[J]. Hereditas, 34(3): 260-268. (in Chinese)
[16] Esteban, M. A. , Wang, T. , Qin, B. M. , Yang, J. Y. , Qin, D. J. , & Cai, J. L. , et al. (2010). Vitamin c enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 6(1): 71-79.
[17] Nakagawa, M. , Koyanagi, M. , Tanabe, K. , Takahashi, K. , Ichisaka, T. , & Aoi, T. , et al. (2008). Generation of induced pluripotent stem cells without myc from mouse and human fibroblasts. Nature Biotechnology, 26(1): 101-106.
[18] Pera, M. F. , & Hasegawa, K. . (2008). Simpler and safer cell reprogramming. Nature Biotechnology, 26(1): 59-60.
[19]王聪慧,赵帅,张译元,等.电穿孔转染制备绵羊诱导多潜能干细胞条件的优化[J].西北农业学报,2015, 24(9): 9-15.
WANG Cong-hui, ZHAO Shuai, ZHANG Yi-yuan, et al. (2015). Optimization of inducing pluripotent stem cells by 0; electroporation in sheep [J]. Acta Agriculturae Boreali-occidentalis sinica, 24(9): 9-15. (in Chinese)
[20] Dattena, M. , Chessa, B. , Lacerenza, D. , Accardo, C. , & Cappai, P. . (2006). Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts. Molecular Reproduction and Development, 73(1): 31-39.
[21] Chin, M. H. , Mason, M. J. , Xie, W. , Volinia, S. , Singer, M. , & Peterson, C. , et al. (2009). Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell, 5(1):111-123.
[22]朱志明,陈红萍,林如龙,等.山麻鸭开产期和产蛋高峰期卵巢组织转录组分析[J].中国农业科学,2016, 49(5): 998-1 007.
ZHU Zhi-ming, CHEN Hong-ping, LIN Ru-long, et al. (2016). Transcriptome analysis of ovary tissue in early laying period and egg laying peak period of Shanma ducks [J]. Scientia Agricultura Sinica, 49(5): 998-1,007. (in Chinese)
[23] Bao, X. , Wu, H. , Zhu, X. , Guo, X. , Hutchins, A. P. , & Luo, Z. , et al. (2015). The p53-induced lincrna-p21 derails somatic cell reprogramming by sustaining h3k9me3 and cpg methylation at pluripotency gene promoters. Cell Research, 25(1): 80-92.
[24] Shi H, Fu Q, Li G, Ren Y, Hu S, Ni W, Guo F, Shi M, Meng L, Zhang H, Qiao J, Guo Z, Chen C. (2015). Roles of p53 and asf1a in the reprogramming of sheep kidney cells to pluripotent cells. Cellular Reprogramming, 17(6): 441-452.
[25] Lee, D. F. , Su, J. , Ang, Y. S. , Carvajalvergara, X. , Muleronavarro, S. , & Pereira, C. F. , et al. (2012). Regulation of embryonic and induced pluripotency by aurora kinase-p53 signaling. Cell Stem Cell, 11(2): 179-194.

绵羊iPS细胞诱导及转录组学分析

Induction and Transcriptomics Analysis of Ovine iPS Cells

PDF下载

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	韦伟, 单守明, 徐文娣, 李光宗. 山葡萄‘双优’组织培养生根期愈伤组织的转录组分析[J]. 新疆农业科学, 2023, 60(6): 1451-1459.
[2]	雷艳, 兰斌, 余万里, 戴小华, 蔡鹏, 顾伟芳, 阿迪莱·艾力, 赵红琼. 缺氧诱导因子和促红细胞生成素及其受体基因在绵羊各组织中表达量[J]. 新疆农业科学, 2023, 60(2): 485-492.
[3]	洪文娟, 侯晨曦, 何宗龙, 决肯·阿尼瓦什. 不同毛色巴什拜羊皮肤组织中LEF1、YWHAZ及WNT2基因差异表达[J]. 新疆农业科学, 2022, 59(11): 2749-2757.
[4]	郭延华, 皮文辉. 用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因[J]. 新疆农业科学, 2021, 58(9): 1747-1755.
[5]	张俊瑜, 郭同军, 桑断疾, 王文奇, 张志军, 王承敏. 饲粮中不同棉秆比例对绵羊瘤胃发酵参数和血清生化指标的影响[J]. 新疆农业科学, 2021, 58(2): 383-392.
[6]	巴·布日格旦, 雒秋江, 谢文龙, 潘榕. 玉米浸泡和粉碎对绵羊日粮消化的影响[J]. 新疆农业科学, 2021, 58(11): 2133-2138.
[7]	古再丽努尔·艾麦提, 郭同军, 张俊瑜, 张志军, 桑断疾, 李聪年. 日粮中不同水平棉秆对育肥期绵羊瘤胃发酵参数和屠宰性能的影响[J]. 新疆农业科学, 2020, 57(3): 581-588.
[8]	许行浩, 侯宇, 赵树林, 杨阳, 余万里, 阿迪莱·艾力, 孙小燕, 赵红琼. 饲喂荨麻干草对羊胃肠动力的影响[J]. 新疆农业科学, 2019, 56(5): 964-971.
[9]	吴红岩, 郭同军, 张志军, 张俊瑜, 桑断疾, 臧长江. 秸秆配合颗粒饲料peNDF水平对绵羊血液生化指标的影响[J]. 新疆农业科学, 2019, 56(4): 749-757.
[10]	郭延华, 万鹏程, 刘长彬. 肉用绵羊超数排卵效果实例分析[J]. 新疆农业科学, 2017, 54(8): 1550-1558.
[11]	于常江;祁成年;张云生;沈敏;杨华;杨永林. 绵羊eIF3h基因克隆、原核表达及蛋白鉴定[J]. , 2017, 54(2): 386-392.
[12]	郭延华, 刘长彬, 万鹏程, 倪建宏, 王立民, 周平, 杨永林, 方志忠, 顾宏伟, 付炳哲, 卢全胜. 肉羊胚胎移植成活的影响因素实例分析[J]. 新疆农业科学, 2017, 54(11): 2138-2145.
[13]	包秋娟, 张丽丽, 海那尔·乌拉孜巴依, 张富春. 干旱胁迫棉花转录组DNA损伤修复相关基因的分析[J]. 新疆农业科学, 2017, 54(11): 1999-2005.
[14]	张译元;唐红;郭延华;王新华;王立民;周平. 绵羊血清蛋白双向凝胶电泳技术的建立[J]. , 2017, 54(1): 190-196.
[15]	沈春修. 水稻LOC_Os10g05490位点冷胁迫条件下表达分析及CRISPR/Cas9定向编辑[J]. 新疆农业科学, 2017, 1(1): 1-10.