牦牛六个多能性相关转录因子OSKMNL的克隆和多顺反子慢病毒载体的构建

doi:10.11843/j.issn.0366-6964.2024.04.021

Abstract

Abstract: The purpose of this study was to clone 6 pluripotency-related transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28 (OSKMNL) of yak and construct polycistronic lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry. In this study, the reproductive crest tissue of a 3-5-month-old healthy female yak fetus was used as the research material. The complete coding region sequences of 6 pluripotent related transcription factors OSKMNL were cloned by RT-PCR and analyzed by bioinformatics. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed by seamless cloning. The lentivirus was packaged with 293T cells, and the packaged lentivirus was infected with 293T cells and yak fibroblasts. The virus infection was detected by fluorescence expression and RT-PCR technique (the experiment was divided into virus infection group and blank control group, each group had 3 repeats). The results showed that the coding region of cloned yak OSKMNL gene was 1 083, 963, 1 434, 1 320, 903 and 618 bp, respectively. Sequence analysis showed that the amino acid sequence of OSKMNL in yak shared more than 99% homology with cattle, and the homology of Sox2, Klf4, c-Myc with cattle was 100%. The results of evolutionary tree showed that yak had the closest genetic relationship with cattle, zebu cattle and buffalo, but the farthest relationship with mice. Protein structure prediction showed that the 6 transcription factors OSKMNL of yak all had the corresponding protein functional structures of the gene family, such as POU domain, HOX domain, HMG domain, Znf-C2H2 domain, HLH domain, CSP domain and so on. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed. 293T cells infected by lentivirus were found to express red and green fluorescence. Yak fibroblasts infected with lentivirus showed red and green fluorescence in the experimental group, and RT-PCR results showed bands with expected size. In this study, 6 pluripotency related transcription factors OSKMNL of yak were successfully cloned. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed, which contain 3 transcription factors of yak, respectively. Lentivirus could infect yak fibroblasts. It is helpful to promote the application of pluripotent related transcription factor OSKMNL in yak stem cells and prepare for the follow-up study of yak iPSC.

Key words: yak, gene cloning, sequence analysis, lentiviral vector

CLC Number:

S823.85

HUANG Xianpeng, XING Jiayi, BAI Yuanyuan, JIANG Yuting, MA Zhiwei, FU Wei, LAN Daoliang. Cloning of Six Pluripotent Related Transcription Factors OSKMNL in Yak and Construction of Polycistron Lentiviral Vector[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1579-1591.

References

[1] GOSZCZYNSKI D E, NAVARRO M, MUTTO A A, et al. Review:embryonic stem cells as tools for in vitro gamete production in livestock[J]. Animal, 2023, 17(Suppl 1):100828.
[2] TAKAHASHI K, YAMANAKA S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006, 126(4):663-676.
[3] POLESSKAYA A, CUVELLIER S, NAGUIBNEVA I, et al. Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency[J]. Genes Dev, 2007, 21(9):1125-1138.
[4] YU J Y, VODYANIK M A, SMUGA-OTTO K, et al. Induced pluripotent stem cell lines derived from human somatic cells[J]. Science, 2007, 318(5858):1917-1920.
[5] SETTHAWONG P, PHAKDEEDINDAN P, TIPTANAVATTANA N, et al. Generation of porcine induced-pluripotent stem cells from Sertoli cells[J]. Theriogenology, 2019, 127:32-40.
[6] ZHOU M, ZHANG M L, GUO T X, et al. Species origin of exogenous transcription factors affects the activation of endogenous pluripotency markers and signaling pathways of porcine induced pluripotent stem cells[J]. Front Cell Dev Biol, 2023, 11:1196273.
[7] KIMURA K, TSUKAMOTO M, TANAKA M, et al. Efficient reprogramming of canine peripheral blood mononuclear cells into induced pluripotent stem cells[J]. Stem Cells Dev, 2021, 30(2):79-90.
[8] ZHANG Y F, HE Y H, WU P, et al. miR-200c-141 enhances sheep kidney cell reprogramming into pluripotent cells by targeting ZEB1[J]. Int J Stem Cells, 2021, 14(4):423-433.
[9] LIU M N, ZHAO L X, WANG Z X, et al. Generation of sheep induced pluripotent stem cells with defined DOX-inducible transcription factors via piggyBac transposition[J]. Front Cell Dev Biol, 2021, 9:785055.
[10] BESSI B W, BOTIGELLI R C, PIERI N C G, et al. Cattle in vitro induced pluripotent stem cells generated and maintained in 5 or 20% oxygen and different supplementation[J]. Cells, 2021, 10(6):1531.
[11] HAN X P, HAN J Y, DING F R, et al. Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells[J]. Cell Res, 2011, 21(10):1509-1512.
[12] SUMER H, LIU J, MALAVER-ORTEGA L F, et al. NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts[J]. J Anim Sci, 2011, 89(9):2708-2716.
[13] OGOREVC J, OREHEK S, DOVČ P. Cellular reprogramming in farm animals:an overview of iPSC generation in the mammalian farm animal species[J]. J Anim Sci Biotechnol, 2016, 7:10.
[14] WEERATUNGA P, HARMAN R M, VAN DE WALLE G R. Induced pluripotent stem cells from domesticated ruminants and their potential for enhancing livestock production[J]. Front Vet Sci, 2023, 10:1129287.
[15] ZHAO L X, WANG Z X, ZHANG J D, et al. Characterization of the single-cell derived bovine induced pluripotent stem cells[J]. Tissue Cell, 2017, 49(5):521-527.
[16] PILLAI V V, KEI T G, REDDY S E, et al. Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance[J]. Anim Sci J, 2019, 90(9):1149-1160.
[17] LIU J, BALEHOSUR D, MURRAY B, et al. Generation and characterization of reprogrammed sheep induced pluripotent stem cells[J]. Theriogenology, 2012, 77(2):338-346.e1.
[18] TIAN L, SHI S H, HU H Y, et al. Sequence analysis and cloning of Oct4 gene in sheep[J]. Grass-Feeding Livestock, 2019(3):19-24. (in Chinese)
田丽, 时邵辉, 胡虹宇, 等. 绵羊Oct4基因的克隆及序列分析[J]. 草食家畜, 2019(3):19-24.
[19] QIU Q, ZHANG G J, MA T, et al. The yak genome and adaptation to life at high altitude[J]. Nat Genet, 2012, 44(8):946-949.
[20] ZHONG J C, WANG H, CHAI Z X, et al. Excavation and innovative utilization of yak germplasm resources[J]. The Chinese Livestock and Poultry Breeding, 2022, 18(10):22-29. (in Chinese)
钟金城, 王会, 柴志欣, 等. 牦牛种质资源挖掘与创新利用[J]. 中国畜禽种业, 2022, 18(10):22-29.
[21] JIA G X, DING L M, XU S R, et al. Conservation and utilization of yak genetic resources in Qinghai-Tibet plateau:problems and perspectives[J]. Acta Ecologica Sinica, 2020, 40(18):6314-6323. (in Chinese)
贾功雪, 丁路明, 徐尚荣, 等. 青藏高原牦牛遗传资源保护和利用:问题与展望[J]. 生态学报, 2020, 40(18):6314-6323.
[22] NICHOLSON M W, TING C Y, CHAN D Z H, et al. Utility of iPSC-derived cells for disease modeling, drug development, and cell therapy[J]. Cells, 2022, 11(11):1853.
[23] ABOUL-SOUD M A M, ALZAHRANI A J, MAHMOUD A. Induced pluripotent stem cells (iPSCs)-roles in regenerative therapies, disease modelling and drug screening[J]. Cells, 2021, 10(9):2319.
[24] CAO H G, YANG P, PU Y, et al. Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins[J]. Int J Biol Sci, 2012, 8(4):498-511.
[25] LUO R J, CAO S Y. Research progress and application prospect of livestock pluripotent stem cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10):4003-4015. (in Chinese)
罗睿杰, 曹素英. 大家畜多能干细胞的研究进展与应用前景[J]. 畜牧兽医学报, 2023, 54(10):4003-4015.
[26] KANG Y, YE S H, ZHAO Q J, et al. Research progress on protection methods of livestock and poultry genetic resource[J]. China Animal Husbandry & Veterinary Medicine, 2013, 40(11):208-212. (in Chinese)
康晔, 叶绍辉, 赵倩君, 等. 畜禽遗传资源保护方法的研究进展[J]. 中国畜牧兽医, 2013, 40(11):208-212.
[27] WEST F D, TERLOUW S L, KWON D J, et al. Porcine induced pluripotent stem cells produce chimeric offspring[J]. Stem Cells Dev, 2010, 19(8):1211-1220.
[28] FAN N N, CHEN J J, SHANG Z C, et al. Piglets cloned from induced pluripotent stem cells[J]. Cell Res, 2013, 23(1):162-166.
[29] VARZIDEH F, GAMBARDELLA J, KANSAKAR U, et al. Molecular mechanisms underlying pluripotency and self-renewal of embryonic stem cells[J]. Int J Mol Sci, 2023, 24(9):8386.
[30] MAKLAD A, SEDEEQ M, CHAN K M, et al. Exploring Lin28 proteins:unravelling structure and functions with emphasis on nervous system malignancies[J]. Life Sci, 2023, 335:122275.
[31] NONG W. Study on Bovine induced pluripotent stem cells[D]. Nanning:Guangxi University, 2014. (in Chinese)
农微. 牛诱导性多能干细胞的初步研究[D]. 南宁:广西大学, 2014.
[32] DENG Y F. Studies on buffalo induced pluripotent stem cells[D]. Nanning:Guangxi University, 2012. (in Chinese)
邓彦飞. 水牛诱导多能干细胞的研究[D]. 南宁:广西大学, 2012.
[33] BOYER L A, LEE T I, COLE M F, et al. Core transcriptional regulatory circuitry in human embryonic stem cells[J]. Cell, 2005, 122(6):947-956.
[34] GHALEB A M, YANG V W. Krüppel-like factor 4(KLF4):what we currently know[J]. Gene, 2017, 611:27-37.
[35] PARK C S, LEWIS A, CHEN T, et al. Concise review:regulation of self-renewal in normal and malignant hematopoietic stem cells by Krüppel-like factor 4[J]. Stem Cells Transl Med, 2019, 8(6):568-574.
[36] FAN W, LI X L. The SIRT1-c-Myc axis in regulation of stem cells[J]. Front Cell Dev Biol, 2023, 11:1236968.
[37] VARLAKHANOVA N V, COTTERMAN R F, DEVRIES W N, et al. myc maintains embryonic stem cell pluripotency and self-renewal[J]. Differentiation, 2010, 80(1):9-19.
[38] FARZANEH M, ATTARI F, KHOSHNAM S E. Concise review:LIN28/let-7 signaling, a critical double-negative feedback loop during pluripotency, reprogramming, and tumorigenicity[J]. Cell Reprogram, 2017, 19(5):289-293.
[39] WU T, WU J, JIN S X, et al. The study on the transfection efficiency of cationic liposome lipofectamine 3000 in adhesive cells, suspension cells and primary cells[J]. Current Immunology, 2018, 38(2):89-94. (in Chinese)
吴婷, 吴江, 金书欣, 等. 阳离子脂质体Lipofectamine 3000对贴壁细胞、悬浮细胞和原代细胞转染效率比较的研究[J]. 现代免疫学, 2018, 38(2):89-94.
[40] DENG Y F, LIU Q Y, LUO C, et al. Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors[J]. Stem Cells Dev, 2012, 21(13):2485-2494.
[41] ZHANG H, CAO S L, LIU S D. Application progress of lentivirus vector system[J]. Chinese Journal of Veterinary Science, 2021, 41(6):1200-1203, 1218. (in Chinese)
张慧, 曹胜亮, 刘思当. 慢病毒载体系统研究及应用进展[J]. 中国兽医学报, 2021, 41(6):1200-1203, 1218.
[42] WANG X Y, MA C C, RODRÍGUEZ LABRADA R, et al. Recent advances in lentiviral vectors for gene therapy[J]. Sci China Life Sci, 2021, 64(11):1842-1857.

Cloning of Six Pluripotent Related Transcription Factors OSKMNL in Yak and Construction of Polycistron Lentiviral Vector

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SHANG Kaiyuan, JIANG Mingfeng, GUAN Jiuqiang, AN Tianwu, ZHAO Hongwen, BAI Qin, WU Weisheng, LI Huade, XIE Rongqing, SHA Quan, LUO Xiaolin, ZHANG Xiangfei. Effects of Maternal Nutritional Regulation in Transition Period on Growth and Development, Serum Biochemistry and Immune Function of Yak Calves [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1638-1648.
[2]	JIANG Jinxiu, ZHANG Jingpeng, LIN Yusheng, LIU Weiwei, HU Qilin, WAN Chunhe. Establishment of a TaqMan Assay for Mycoplasma ovipneumoniae based on the Hsp70 Gene and Analysis of Its Genetic Evolution [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1684-1695.
[3]	ZUO Zizhen, WANG Haibo, CHAI Zhixin, FU Jianhui, ZHANG Xiangfei, LUO Xiaolin, ZHONG Jincheng. Effects of Rumen-protected Methionine on Meat Quality, Volatile Flavor Compounds and Fatty Acid Composition of Yak Semitendinosus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1102-1114.
[4]	LIU Bin, WANG Meng, PAN Yangyang, WANG Jinglei, XU Gengquan. Effect of LPA on the Expression of HAS2, PTGS2 and PTX3 in Cumulus Cells of Yak (Bos grunniens) [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 552-561.
[5]	ZHANG Jinpeng, CHEN Cuiteng, LIN Lin, FU Huanru, LI Zhaolong, JIANG Bin, HUANG Yu, WAN Chunhe. Establishment of a Real-time Fluorescent RT-PCR Assay for Detection of Pigeon Megrivirus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 860-866.
[6]	LIU Yili, TANG Jiao, MIN Qi, YANG Lu, WANG Zening, HU Lian, ZHAO Di, JIANG Mingfeng. Mining Key Candidate Genes of Development and Metabolism in Yak Abomasum Based on Transcriptome Data [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 153-168.
[7]	YAO Ying, ZHOU Yingcong, DU Peiyan, LI Yijuan, QIAN Wenjie, LI Liuyang, YU Zhipeng, CUI Yan, YU Sijiu, FAN Jiangfeng. Proteomic Analysis of Yak Serum During Pregnancy Based on TMT Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 192-206.
[8]	ZHANG Ying, YUAN Ligang, CHEN Guojuan, ZHANG Fang, YANG Dapeng. Analysis of Differential Expression of ET-1/eNOS in the Development of Cryptorchidism in Yak [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 207-217.
[9]	LI Zaiwen, LI Xiang, LI Xiaowei, LI Biao, JIANG Mingfeng. The Pedigree Reconstruction of the Maiwa Yak Preserved Population Based on GBS Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3710-3721.
[10]	WANG Jingyu, PAN Yangyang, XU Gengquan, ZHANG Rui, ZHANG Wenlan, WANG Xiaoshan, WU Rentaodi, ZHAO Rigetu, CUI Yan, YU Sijiu. Preparation and Preliminary Application of Yak (Bos Grunniens) Fas-associated Factor 1 Polyclonal Antibody [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3369-3382.
[11]	ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.
[12]	SHAO Yuexin, ZHANG Xinyu, GE Liyan, SHI Huaiping. Cloning and RNA Interference Analysis of ATF4 Gene in Xinong Saanen Dairy Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2353-2364.
[13]	JIAO Zhengxing, PAN Yangyang, WANG Meng, WANG Jinglei, MA Wenbin, GAO Xiang, ZHANG Hui, CUI Yan, YU Sijiu, WANG Libin. Preparation of Polyclonal Antibody against Yak LC3B Protein and Its Application in Detection of Expression in Reproductive Organs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2436-2447.
[14]	CAO Liyan, KONG Xiangyu, LI Xiangtong, SUO Xuepeng, DUAN Yueyue, YUAN Cong, SHI Lei, ZHANG Yu, MA Guoxiang, ZHENG Haixue, WANG Qi. Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2487-2497.
[15]	CHANG Yiming, TANG Cheng, YUE Hua. Molecular Epidemiological Investigation of Bovine Respiratory Syncytial Virus in Yaks [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2030-2041.