Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (17): 3566-3575.doi: 10.3864/j.issn.0578-1752.2012.17.013

• ANIMAL SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Molecular Cloning and Application in Cell Reprogramming rom Porcine L-Myc Gene

 LI  Zhen-Zhen, CUI  Yi, GAO  Yi, CHENG  De, LIU  Ya-Jun, WANG  Hua-Yan   

  1. 1.西北农林科技大学动物医学院/陕西省干细胞工程技术研究中心,陕西杨凌 712100
  • Received:2012-02-27 Online:2012-09-01 Published:2012-04-28

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

[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.
[1] CHEN Rui-ji,GAO Xiang,CHEN Qi-jiao,DONG Jian,ZHAO Wan-chun,LI Yan-liang,WANG Ming-xia,LI Min,PANG Hong-xi,LI Zhe-qing,LIU Jun
.

Cloning and Prokaryotic Expression of an avenin-like from Wheat Variety Shaan 253

[J]. Scientia Agricultura Sinica, 2010, 43(9): 1954-1962 .
[2] LI Min,GAO Xiang,CHEN Qi-jiao,DONG Jian,ZHAO Wan-chun,WANG Ming-xia
. Cloning, Prokaryotic Expression and In vitro Functional Analysis of α-Gliadin Gene from Common Wheat
[J]. Scientia Agricultura Sinica, 2010, 43(23): 4765-4774 .
[3] WANG Kai,TIAN Wen-ru,GAO Shan-song
. Enhancement of Heat Tolerance in Mice Early Embryos by TCZB-Culture Medium#br# [J]. Scientia Agricultura Sinica, 2010, 43(13): 2800-2805 .
[4] . Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and its Expression [J]. Scientia Agricultura Sinica, 2007, 40(2): 373-378 .
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!