猪L-Myc基因的分子克隆及在细胞重编程中的应用

doi:10.3864/j.issn.0578-1752.2012.17.013

摘要/Abstract

摘要： 【目的】克隆猪L-Myc基因，并在蛋白水平表达的情况下探索其在细胞重编程中的作用，为深入研究猪L-Myc基因替代c-Myc诱导多能干细胞（induced pluripotent stem cell，iPSC）奠定基础。【方法】先通过NCBI序列比对，采用RT-PCR克隆猪L-Myc基因cDNA，生物信息学分析猪L-Myc基因与人和小鼠的同源性，构建融合表达载体pEGFP/L-Myc-C1，通过载体转染和免疫印记检测猪L-Myc基因cDNA的蛋白水平表达；再将L-Myc基因装入逆转录病毒载体中，分别使用不同的转录因子诱导猪胎儿成纤维细胞（porcine embryo fibroblast，PEF），通过形态变化和碱性磷酸酶（AP）染色验证猪L-Myc基因在细胞重编程中的作用。【结果】①获得了1 113 bp的猪L-Myc基因cDNA，编码364个氨基酸，理论分子质量为40 kD；②生物信息学分析显示猪L-Myc基因与人和小鼠高度同源；③免疫印记检测结果说明猪L-Myc基因cDNA能够在蛋白水平表达；④细胞诱导试验和AP染色结果显示转录因子Oct4、Sox2、Klf4和L-Myc（OSKL）组合诱导的细胞阳性克隆率明显高于Oct4、Sox2和Klf4（OSK）组合的阳性克隆率。【结论】获得了猪L-Myc基因，并且该基因在蛋白水平表达且在细胞重编程过程中起到了重要的作用。

关键词: 猪L-Myc基因, 诱导多能干细胞（iPSC）, 原癌基因, 致瘤性, 融合蛋白

Abstract: 【Objective】Cloning porcine L-Myc gene and exploring its function in cell reprogramming at the protein level could be helpful to provide a foundation for substituting L-Myc gene for c-Myc gene in the induction of induced pluripotent stem (iPS) cells in porcine. 【Method】Through NCBI sequence alignment, porcine L-Myc gene cDNA was obtained by RT-PCR. The homology of L-Myc gene among these three species of porcine, human and mouse was analyzed by bioinformatics. The fusion expression vector pEGFP/L-Myc-C1 was constructed, and the vector-transfected and western blotting were used to detect the expression of the cloned porcine L-Myc gene cDNA at the protein level. Then the L-Myc gene was inserted into the retroviral vector, and different transcription factors, respectively, induced porcine embryo fibroblast (PEF) cells. Through observing the variation of the induced cells and alkaline phosphatase (AP) staining, the important role porcine L-Myc gene plays in cell reprogramming was verified. 【Result】 A 1 113 bp porcine L-Myc gene cDNA sequence was obtained by RT-PCR, encoding 364 amino acids and the theoretical molecular weight is 40 kD. Bioinformatics analysis showed that a high degree of homology in L-Myc existed among porcine, human and mouse. The western blotting analysis proved that the cloned porcine L-Myc gene cDNA could be expressed at the protein level. After induction, the morphology of induced cells are varied, and the quantity of positive clones induced with the transcription factors Oct4, Sox2, Klf4, and L-Myc (OSKL) was far more than that with Oct4, Sox2, and Klf4 (OSK). 【Conclusion】 The porcine L-Myc, a transcription factor, was cloned, and the results of analysis showed that porcine L-Myc gene plays an important role in cell reprogramming process.

Key words: porcine L-Myc gene, induced pluripotent stem cell (iPSC), protooncogene, tumorigenic, fusion protein

李珍珍, 崔怡, 高祎, 成德, 刘亚军, 王华岩. 猪L-Myc基因的分子克隆及在细胞重编程中的应用[J]. 中国农业科学, 2012, 45(17): 3566-3575.

LI Zhen-Zhen, CUI Yi, GAO Yi, CHENG De, LIU Ya-Jun, WANG Hua-Yan. Molecular Cloning and Application in Cell Reprogramming rom Porcine L-Myc Gene[J]. Scientia Agricultura Sinica, 2012, 45(17): 3566-3575.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.17.013

https://www.chinaagrisci.com/CN/Y2012/V45/I17/3566

参考文献

[1]Nakagawa M, Takizawa N, Narita M, Ichisaka T, Yamanaka S. Promotion of direct reprogramming by transformation-deficient Myc. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(32): 14152-14157.

[2]Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676.

[3]Okita K, Ichisaka T, Yamanaka S. Generation of germ-line competent induced pluripotent stem cells. Nature, 2007, 448(7151): 313-317.

[4]Takahashi K, Tanabe K, Narita M O M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, 131(5): 861-872.

[5]Luscher B, Eisenman R N. New light on Myc and Myb. Part I. Myc. Genes and Development, 1990, 4(12A): 2025-2035.

[6]Sheiness D, Bishop J M. DNA and RNA from uninfected vertebrate cells contain nucleotide sequences related to the putative transforming gene of avian myelocytomatosis virus. Journal of Virology, 1979, 31(2): 514-521.

[7]DePinho R A, Leguoy E, Feldman L B, Kohl N E, Yancopoulos G D, Alt F W. Structure and expression of the murine N-myc gene. Proceedings of the National Academy of Sciences of the United States of America, 1986, 83(6): 1827-1831.

[8]Kohl N E, Leguoy E, DePinho R A, Nisen P D, Smith R K, Gee C E, Alt F W. Human N-myc is closely related in organization and nucleotide sequence to c-myc. Nature, 1986, 319(6048): 73-77.

[9]Schwab M, Alitalo K, Klempnauer K H, Varmus H E, Bishop J M, Gilbert F, Brodeur G, Goldstein M, Trent J. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumor. Nature, 1983 305(5931): 245-248.

[10]Kaye F, Battey J, Nau M, Brooks B, Seifter E, de Greve J, Birrer M, Sausville E, Minna J. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing. Molecular and Cell Bioloyg, 1988, 8(1): 186-195.

[11]Nau M M, Brooks B J, Batey J, Sausville E, Gazdar A F, Kirsch I R, McBride O W, Bertness V, Hollis GF, Minna J D. L-myc, a new myc-related gene amplified and expressed in human small cell lung carcinomas. Nature, 1985, 318(6041): 69-73.

[12]DePinho R A, Hatton K S, Tesfaye A, Vancopoulos G D, Alt F W. The human myc gene family: structure and activity of L-myc and an L-myc pseudogene. Genes and Development, 1987, 1(10): 1311-1326.

[13]Leguoy E, DePinho R, Zimmerman K, Collum R, Yancopoulos G, Mitsock L, Kriz R, Alt F W. Structure and expression of the murine L-myc gene. The EMBO Journal, 1987, 6(11): 3359-3366.

[14]Malynn B A, de Alboran IM, O’Hagan R C, Bronson R, Davidson L, DePinho R A, Alt F W. N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. Genes and Development, 2000, 14(11): 1390-1399.

[15]Birrer M J, Segal S, DeGreve J S, Kaye F, Sausville E A, Minna J D. L-myc cooperates with ras to transform primary rat embryo fibroblasts. Molecular and Cell Biology, 1988, 8(6): 2668-2673.

[16]Oster S K, Mao D Y, Kennedy J, Penn L Z. Functional analysis of the N-terminal domain of the Myc oncoprotein. Oncogene, 2003, 22(13):1998-2010.

[17]Hatton K S, Mahon K, Chin L, Chiu F C, Lee H W, Peng D, Morgenbesser S D, Horner J, DePinho R A. Expression and activity of L-Myc in normal mouse development. Molecular and Cell Biology, 1996, 16(4): 1794-1804.

[18]Barrett J, Birrer M J, Kato G J, Dosaka-Akita H, Dang C V. Activaion domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells. Molecular and Cell Biology, 1992, 12(7): 3130-3137.

[19]Cole M D, Cowling V H. Transcription-independent functions of MYC: regulation of translation and DNA replication. Nature Reviews Molecular Cell Biology, 2008, 9(10): 810-815.

[20]Schwab M, Varmus H E, Bishop J M. Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature, 1985, 316(6024): 160-162.

[21]Yancopoulos G D, Nisen P D, Tesfaye A, Kohl N E, Goldfarb M P, Alt F W. N-myc can cooperate with ras to transform normal cells in culture. Proceedings of the National Academy of Sciences of the United States of America, 1985, 82(16): 5455-5459.

[22]Mario´n R M, Strati K, Li H, Murga M, Blanco R, Ortega S, Fernandez-Capetillo O, Serrano M, Hlasco M A. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature, 2009, 460(7259): 1149-1153.

[23]Kawamura T, Suzuki J, Wang Y Y, Menendez S, Morera L B, Raya A, Wahl G M, Belmonte J C I. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature, 460(7259): 1140-1144.

[24]Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K, Yamanaka S. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature, 2009, 460(7259): 1132-1135.

[25]Zhao Y, Yin X L, Qin H, Zhu F F, Liu H S, Yang W F, Zhang Q, Xiang C G, Hou P P, Song Z H, Liu Y X, Yong J, Zhang P B, Cai J, Liu M, Li H G, Li Y Q, Qu X X, Cui K, Zhang W Q, Xiang T T, Wu Y T, Zhao Y D, Liu C, Yu C, Yuan K, Lou J J, Ding M X, Deng H K. Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell, 2008, 3(5): 475-479.

[26]Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology, 2008, 26(1): 101-106.

[27]Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008, 322(5903): 949-953.

[28]Yusa K, Rad R, Takeda J, Bradley A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nature Methods, 2009, 6(5): 363-369.

[29]Woltjen K, Michael I P, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung H K, Nagy A. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 2009, 458(7239): 766-770.

[30]Kim D H, Kim C H, Moon J I, Chung Y G, Chang M Y, Han B S, Ko S, Yang E, Cha K Y, Lanza R, Kim K S. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell, 2009, 4(6): 472-476.

[31]Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson J A. Human induced pluripotent stem cells free of vector and transgene sequences. Science, 2009, 324(5928): 797-801.

[32]Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy. Series B Physical and Biological Sciences, 2009, 85(8): 348-362.

[33]Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem cells generated without viral integration. Science, 2008, 322(5903): 945-949.

[34]Zhou W, Freed C R. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells, 2009, 27(11): 2667-2674.

[35]Zhou H, Wu S, Joo J Y, Zhu S, Han D W, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Schöler H R, Duan L, Ding S. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 2009, 4(5): 381-384.