徐淮山羊多能性转录因子mRNA制备及在成纤维细胞中的表达

doi:10.3864/j.issn.0578-1752.2014.07.019

摘要/Abstract

摘要： 【目的】克隆徐淮山羊多能性转录因子Oct4、Sox2、Klf4和c-Myc的CDS片段，构建pMD19-T-oct4、pMD19-T-sox2、pMD19-T-klf4和pMD19-T-c-myc重组质粒，随后构建含T7启动子的pcDNA3-oct4、pcDNA3-sox2、pcDNA3-klf4和pcDNA3-c-myc重组质粒，通过体外转录，获得该4种多能性转录因子的mRNA，并使其在徐淮山羊成纤维细胞中稳定表达。【方法】应用RT-PCR方法分别从徐淮山羊睾丸组织、皮肤组织、小肠组织中扩增多能性转录因子Oct4、Sox2、Klf4和c-Myc基因编码全序列，将该4种基因分别克隆到pMD19-T载体，构建pMD19-T-oct4、pMD19-T-sox2、pMD19-T-klf4和pMD19-T-c-myc重组质粒。然后将pcDNA3.0载体和pMD19-T重组载体双酶切后用T4连接酶连接，构建含有T7启动子的真核表达载体pcDNA3-oct4、pcDNA3-sox2、pcDNA3-klf4和pcDNA3-c-myc重组质粒。各重组质粒分别用限制性内切酶XhoI、XbaI单酶切，酶切后质粒模版按照体外转录试剂盒说明体外转录获得各多能性转录因子的mRNA，并对获得的mRNA进行检测，确定其稳定性和浓度。按照脂质体（体积）﹕mRNA（质量）为1﹕1的比例用脂质体2000转染。转染24 h后，利用Western blot技术、间接免疫荧光实验检测徐淮山羊多能性转录因子Oct4、Sox2、Klf4和c-Myc基因的mRNA在成纤维细胞中的表达。【结果】①克隆得到的徐淮山羊Oct4、Sox2、Klf4和c-Myc基因编码序列全长分别为1 083、962、1 434和1 320 bp，并经TA克隆测序验证，其CDS 序列与绵羊、人、牛和猪等的序列相似性在89%以上；②体外转录获得的4种多能性转录因子的mRNA经脂质体转染徐淮山羊成纤维细胞，在成纤维细胞中均定位于细胞核；③4种多能性转录因子的mRNA在徐淮山羊成纤维细胞中表达的蛋白与预期大小一致，分别为38、34、50和48 kd。【结论】成功克隆了徐淮山羊Oct4、Sox2、Klf4和c-Myc基因，该4种多能性转录因子的mRNA能够在徐淮山羊成纤维细胞中稳定表达，为进一步研究Oct4、Sox2、Klf4和c-Myc基因的功能和山羊体细胞的重编程奠定基础。

关键词: 徐淮山羊 , 多能性转录因子 , 体外转录 , mRNA

Abstract: 【Objective】 The genes of Oct4, Sox2, Klf4 and c-Myc were cloned, the pMD19-T-oct4, pMD19-T-sox2, pMD19-T-klf4, and pMD19-T-c-myc recombinant plasmids were constructed, and then the pcDNA3-Oct4, pcDNA3 -Sox2, pcDNA3-Klf4 and pcDNA3-c-Myc recombinant plasmids containing T7 promoter were constructed. According to the in vitro transcription, the four mRNA transcription factors were obtained, and it was made to express stably in Xuhuai goat fibroblasts. 【Method】Through the testicular, skin and small intestine tissue, the Oct4, Sox2, Klf4, and c-Myc genes coding sequence were obtained by RT-PCR, and were cloned into pMD19-T vector, and then cloned into the eukaryotic expression pCDNA3.0 vector that contains T7 promoter, and then the pcDNA3-Oct4, pcDNA3-Sox2, pcDNA3-Klf4 and pcDNA3-c-Myc recombinant plasmids were constructed. Recombinant plasmids were linearized using XhoI and XbaI, in vitro transcription of the transcription factors was made to obtain the mRNA using the instructions of in vitro transcription, the mRNA was detected, its stability and concentration were determined, Lipofectamine 2000 transfection was carried out according to the proportion of Lipofectamine﹕mRNA=1﹕1. Western blot technique and immunofluorescence assay were used to detect the mRNA expression in Xuhuai goat fibroblasts 24 h post-transfection. 【Result】 The length of Xuhuai goat Oct4, Sox2, Klf4 and c-Myc genes coding sequence were 1 083 bp, 962 bp, 1 434 bp and 1 320 bp, and confirmed by TA cloning, the sequences similarity of coding sequences of sheep, human, cattle and pig were above 89%. The mRNA of four transcription factors by transfection to Xuhuai goat fibroblasts were localized in the nucleus. The mRNA of four transcription factors expression in fibroblasts proteins was consisted with the expected size, 38 kd, 34 kd, 50 kd and 48 kd. 【Conclusion】The Xuhuai goat Oct4, Sox2, Klf4 and c-Myc transcription factors were cloned successfully, and the four mRNA can express in fibroblasts stably, thus a foundation for further study of the function of the genes Oct4, Sox2, Klf4, c-Myc and goat somatic cell reprogramming is established.

Key words: Xuhuai goat , transcription factors , in vitro transcription , mRNA

邱峰龙, 张亚妮, 倪蓉, 李伟, 陈庭锋, 韦光辉, 李碧春. 徐淮山羊多能性转录因子mRNA制备及在成纤维细胞中的表达[J]. 中国农业科学, 2014, 47(7): 1417-1426.

QIU Feng-Long, ZHANG Ya-Ni, NI Rong, LI Wei, CHEN Ting-Feng, WEI Guang-Hui, LI Bi-Chun. The Preparation of mRNA Pluripotency Transcription Factors and Its Expression in Xuhuai Goat Fibroblasts[J]. Scientia Agricultura Sinica, 2014, 47(7): 1417-1426.

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.0578-1752.2014.07.019

https://www.chinaagrisci.com/CN/Y2014/V47/I7/1417

参考文献

[1]Schmidt E V. The role of c-myc in cellular growth control. Oncogene, 1999, 18(19): 2988-2996.

[2]Warren L, Manos P D, Ahfeldt T L, Loh Y H, Li H, Lau F, Ebina W, Mandal P K, Smith Z D, Meissner A, Daley G Q, Brack A S, Collins J J, Cowan C, Schlaeger T M, Rossi D J. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010, 7(5): 618-630.

[3]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.

[4]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.

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

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

[7]张金吨, 赵丽霞, 吴宝江, 李云霞, 张静南, 苏杰, 孙伟, 戴雁峰, 郭继彤, 胡树香, 李瑶, 汪玮, 刘鹏涛, 李喜和. 利用 piggyBac 载体制备小鼠诱导性多能干细胞. 中国科学: 生命科学, 2012, 42(7): 553-561.

Zhang J D, Zhao L X, Wu B J, Li Y X, Zhang J N, Su J, Sun W, Dai Y F, Guo J T, Hu S X, Li Y, Wang W, Liu P T, Li X H. Mouse induced pluripotent stem cells generated by piggyback. Scientia Sinica Vitae, 2012, 42(7): 553-561. (in Chinese)

[8]宋成义, 周家庆, 冯晓军, 谢雨琇, 李庆平, 吴晗, 高波, 王霄燕. 猪CatSperB 和CatSperG 基因的克隆, 表达及生物信息学. 中国农业科学, 2012, 45(21): 4465-4474.

Song C Y, Zhou J Q, Feng X J, Xie Y X, Li Q P, Wu H, Gao B, Wang X Y. Cloning, expression and bioinformatics analysis of porcine CatSperB and CatSperG genes. Scientia Agricultura Sinica, 2012, 45(21): 4465-4474. (in Chinese)

[9]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. 中国农业科学, 2012, 45(17): 3566-3575.

Li Z Z, Cui Y, Gao Y, Cheng D, Liu Y J, Wang H Y. Molecular cloning and application in cell reprogramming from porcine L-Myc gene. Scientia Agricultura Sinica, 2012, 45(17): 3566-3575. (in Chinese)

[10]Medvedve S P, Shevchenko A I, Mazurok N A, Zakiian S M. Oct4 and NANOG are the key genes in the system of pluripotency maintenance in mammalian cell. Genetika, 2008, 44(12): 1589-1608.

[11]Kirchhof N, Carnwath J W, Lemme E, Anastassiadis K, Schler H, Niemann H. Expression pattern of Oct-4 in preimplantation embryos of different species. Biology of Reproduction, 2000, 63(6): 1698-1705.

[12]Cheng L, Sung M T, Cossu-Rocca P, Jones T D, Maclennan G T, Jong J D, Lopez-beltran A, Montironi R, Looijenga L H J. OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. Journal of Pathology, 2007, 211(1): 1-9.

[13]Pesce M, Scholer H. Oct4: control of totipotency and germline deternmination. Molecular Reproduction and Development, 2000, 55: 452-457.

[14]Kues W A, Peterseb B, Mysegades W, Joseph W, Carnwath Heiner N. Isolation of murine and porcin fetal stem cells from somatic tissue. Biology of Reproduction, 2005, 72(4): 1020-1028.

[15]Boiani M, Scholer H R. Regulatory networks in embryo-derived pluripotent stem cells. Nature Review Molecular Cell Biology, 2005, 6(11): 872-884.

[16]Avilion A A, Nicolis S K, Pevny L H, Perez L, Vivian N, Lovell-badge R. Multipotent cell lineages in early mouse development depeng on SOX2 function. Genes Development, 2003, 17(1): 126-140.

[17]Masui S, Nakatake Y, Toyooka Y, Daisuke S, Rika Y, Kazue T, Hitoshi O, Akihiko O, Ryo M, Alexei A S, Minoru S H. Pluripotency governed by Sox2 via regulation of Oct3/ 4 ex pression in mouse embryonic stem cells. Nature Cell Biology, 2007, 9(6): 625-635.

[18]Garrett L A, Eberspaecher H, Seldin M F, Crombrugghe B. A gene for a novel zinc-finger protin expressed in differentiated epithelial cells and transiently in certain mesenchymal cells. Journal of Biological Chemistry, 1996, 271(49): 31384-31390.

[19]Katz J P, Perreault N, Goldstein B G, Actman L, Mcnally S R, Silberg D G, Furth E E, Kaestner K H. Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach. Gastroenterology, 2005, 128(4): 935-945.

[20]Foster K W, Frost A R, McKie-Bell P. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Research, 2000, 60(22): 6488-6495.

[21]宁淑芳, 李青洋, 梁明明, 徐惠艳, 杨小淦, 陆阳清, 卢晟盛, 卢克焕. 广西巴马小型猪Oct4基因克隆及其在成纤维细胞中的表达. 中国兽医学报, 2012, 32(4): 501-520.

Ning S F, Li Q Y, Liang M M, Xu H Y, Yang X J, Lu Y Q, Lu S S, Lu K H. Cloning of Guangxi banma minipig Oct4 gene and its expression in fibroblast cells. Chinese Journal of Veterinary Science, 2012, 32(4): 501-520. (in Chinese)

[22]郑喜邦, 杨照海, 刘平, 赵华, 卢晟盛, 卢克焕, 毕方方. 山羊Sox2基因克隆、原核表达和GST融合蛋白纯化. 畜牧兽医学报, 2009, 40(10): 1454-1459.

Zheng X B, Yang Z H, Liu P, Zhao H, Lu S S, Lu K H, Bi F F. Cloning of capra hircus Sox 2, and its expression in escherichia coli and purification of GST-Sox2 fusion protein. Acta Veterinaria et Zootechnica Sinica, 2009, 40(10): 1454-1459. (in Chinese)

[23]王春生, 罗芳, 杜文敬, 安铁洙. 小鼠Klf4基因的克隆及原核表达分析. 中国实验动物学报, 2009, 17(2): 103-107.

Wang C S, Luo F, Du W J, An T Z. Cloning and prokaryotic expression of Krppel-Like Factor 4 in mouse. Acta Laboratorium Animalis Scientia Sinica, 2009, 17(2): 103-107.(in Chinese)

[24]赵华, 杨照海, 刘平, 卢晟盛, 卢克焕, 毕方方, 郑喜邦. 山羊C-myc原癌基因克隆、原核表达和GST-Myc融合蛋白纯化. 农业生物技术学报, 2010, 18(5): 931-937.

Zhao H, Yang Z H, Liu P, Lu S S, Lu K H, Bi F F, Zheng X B. Cloning and prokaryotic expression of goat c-Myc proto-oncogene, and purification of GST-Myc fusion protein. Journal of Agricultural Biotechnology, 2010, 18(5): 931-937. (in Chinese)

[25]Matthew A, Mehmet F Y. Innate immune suppression enables frequent transfection with RNA encoding reorogramming proteins. Plos One, 2010, 5(7): e11756.

[26]Plews J R, Li J L, Mark J, Harry D M, Chris M, Peter W A, Jie N. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach. Plos One, 2010, 5(12): e14397.

[27]Ren J T, Pak Y J, He L Z, Qian L, Gu Y Z, Li H, Rao L J, Liao J, Cui C, Xu X, Zhou J Q, Ri H, Xiao L. Generation of hircine-induced pluripotent stem cells by somatic cell reprogramming. Cell Research, 2011(21): 849-853.