利用诱导因子Oct4、Sox2和SV40 T的mRNA介导体细胞重编程研究

畜牧兽医学报

• 遗传繁育 • 上一篇下一篇

利用诱导因子Oct4、Sox2和SV40 T的mRNA介导体细胞重编程研究

潘传英^1,2,3,陈宏^2*,BISHOP E.Colin³

（1. 西北农林科技大学生命科学学院，杨凌 712100； 2. 西北农林科技大学动物科技学院，杨凌 712100；3. Institute for Regenerative Medicine, Wake Forest University, WinstonSalem, NC 27101, USA)

收稿日期:2012-01-04 出版日期:2012-11-26 发布日期:2012-11-26
通讯作者: 陈宏，E-mail:chenhong1212@263.net
作者简介:潘传英(1980-)，女，四川达县人，讲师，博士, 主要从事细胞生物学、遗传学的研究工作，Tel：029-87092262，E-mail: chuanyingpan@yahoo.com
基金资助:
国家自然科学基金(31000655)；西北农林科技大学博士启动基金 (2010BSJJ049)；NIH 项目(R21 RR025408)

Cellular Reprogramming by in vitro Transcribed mRNA Cocktail of Oct4, Sox2 and SV40 T-antigen

PAN Chuan-ying^1，2，3, CHEN Hong^2*, BISHOP E.Colin³

（1. College of Life Sciences, Northwest A&F University, Yangling 712100, China; 2. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; 3. Institute for Regenerative Medicine, Wake Forest University, WinstonSalem, NC 27101, USA）

Received:2012-01-04 Online:2012-11-26 Published:2012-11-26

摘要/Abstract

摘要： 为利用特定诱导因子Oct4、Sox2和SV40 T的体外转录mRNA实现安全的成纤维细胞重编程，本研究成功构建了特定诱导因子Oct4、Sox2和SV40 T的mRNA体外转录载体，并对体外转录mRNA进行了5′和3′端加工修饰；进行了诱导因子mRNA的293和IMR90细胞转染试验，利用免疫细胞化学、免疫荧光和RealTime PCR分别检测了Oct4、Sox2、SV40 T和Nanog的表达情况。结果显示，以上2种细胞以Oct4∶Sox2∶SV40 T=2∶1∶1 的mRNA比例转染后均表达这3种特定诱导因子，且相应蛋白都正确地定位在细胞核上。转染细胞中Oct4和Nanog的表达都特异性地增高。结果提示，3种诱导因子的体外转录mRNA能够在体内协同作用，激活细胞内源性干性标志基因Nanog的表达，为开启重编程过程奠定了坚实的基础。

Abstract: The aim of this study was to reprogram fibroblasts cell by in vitro transcribed mRNA cocktail of Oct4, Sox2 and SV40 T-antigen, and provide a safe, nonintegrating strategy for somatic cell reprogramming. The recombinated mRNA transcription vectors including the above three induced facotrs genes were constructed and transcripted, respectively, then 5′UTR and 3′ UTR of β-globin gene were used for stabilizing in vitro transcribed mRNA following in vitro capping and poly(A) tailing. After mRNA transfection of 293 and IMR90 cells, immunocytochemistry, immunoflurescence and Real-Time PCR methods were used to detect gene expression, protein location and endogenous gene expression related to pluripotence. The results showed that target gene expressions could be detected in 293 and IMR90 cells and all expressed protein were localized properly in the nucleus. The specific expression of Oct4 and Nanog were increased in transfection cells. Endogenous Nanog expression was induced by mRNA cock-tail transfection. These findings indicate that mRNA of Oct4, Sox2 and SV40 T in vitro can coorperate to initiate cellular reprogramming.

中图分类号:

S813.3
Q2

潘传英,陈宏,BISHOP E.Colin. 利用诱导因子Oct4、Sox2和SV40 T的mRNA介导体细胞重编程研究[J]. 畜牧兽医学报, doi: .

PAN Chuan-ying, CHEN Hong, BISHOP E.Colin. Cellular Reprogramming by in vitro Transcribed mRNA Cocktail of Oct4, Sox2 and SV40 T-antigen[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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参考文献

［1］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.

［2］YU J, VODYANIK M, SMUGA-OTTO K, et al. Induced pluripotent stem cell lines derived from human somatic cells［J］. Science, 2007, 318(5858):1917-1920.

［3］CHAMBERLAIN S, LI X, LALANDE M. Induced pluripotent stem (iPS) cells as in vitro models of human neurogenetic disorders［J］. Neurogenetics, 2008, 9(4):227-235.

［4］DIMOS J, RODALFA K, NIAKAN K, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons［J］. Science, 2008, 321(5893):1218-1221.

［5］PARK I, ZHAO R, WEST J, et al. Reprogramming of human somatic cells to pluripotency with defined factors［J］. Nature, 2008, 451(7175):141-146.

［6］OKITA K, NAKAGAWA M, HYENJONG H, et al. Generation of mouse induced pluripotent stem cells without viral vectors［J］. Science, 2008, 322(5903):949-953.

［7］STADTFELD M, NAGAYA M, UTIKAL J, et al. Induced pluripotent stem cells generated without viral integration［J］. Science, 2008, 322(5903):945-949.

［8］KAJI K, NORRBY K, PACA A, et al. Virusfree induction of pluripotency and subsequent excision of reprogramming factors［J］. Nature, 2009, 458(7239): 771-775.

［9］WOLTJEN K, MICHAEL I P, MOHSENI P, et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells ［J］. Nature, 2009, 458(7239): 766-770.

［10］SOLDNER F, HOCKEMEYER D, BEARD C, et al. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors ［J］. Cell, 2009, 136(5): 964-977.

［11］ZHOU H, WU S, JOO J, et al. Generation of induced pluripotent stem cells using recombinant proteins ［J］. Cell Stem Cell, 2009, 4(5):381-384.

［12］KIM D, KIM C H, MOON J I, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins ［J］. Cell Stem Cell, 2009, 4(6):472-476.

［13］PAN C, HICKS A, CHEN H, et al. SNL fibroblast feeder layers support derivation and mainteence of human induced pluripotent stem cells ［J］. J Genet Genomics, 2010, 37:241-248.

［14］PAN C, LU B, CHEN H, et al. Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins Oct4, Sox2, Klf4 and c-MYC ［J］. Mol Biol Reports, 2010, 37:2117-2124.

［15］PAN C, LAN X, CHEN H, et al. An economical single-sided antibody incubation method for western blotting ［J］. J Virol Methods, 2010, 169:409-411.

［16］RUSSELL J E, LIEBHABER S A. The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3′untranslated region ［J］. Blood, 1996, 87: 5314-5323.

［17］YU J, RUSSELL J E. Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA ［J］. Mol Cell Biol, 2001, 21:5879-5888.

［18］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.

［19］TORRES J, WATT F M. Nanog maintains pluripotency of mouse embryonic stem cells by inhibiting NFkappaB and cooperating with Stat3 ［J］. Nat Cell Biol, 2008, 10(2):194-201.

［20］MATTHEW A, MEHMET F Y. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins ［J］.PLoS One, 2010, 5(7):e11756.

［21］WARREN L, MANOS P D, AHFELDT T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA ［J］. Cell Stem Cell, 2010, 7(5):618-630.

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