Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立

doi:10.16431/j.cnki.1671-7236.2016.01.003

›› 2016, Vol. 43 ›› Issue (1): 16-22.doi: 10.16431/j.cnki.1671-7236.2016.01.003

Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立

刘怀禹^1,2, 李浩天^1,2, 苏小虎^1,2, 孟凡华^1,2, 刘春霞^1,2, 张焱如^1,2, 曹俊伟^1,2

1. 内蒙古农业大学生命科学学院, 呼和浩特 010018;
2. 内蒙古自治区生物制造重点实验室, 呼和浩特 010018

收稿日期:2015-06-23 出版日期:2016-01-20 发布日期:2016-01-23
通讯作者: 张焱如 E-mail:yanru1964@163.com
作者简介:刘怀禹(1989-),男,河北邯郸人,硕士生,研究方向:动物成体干细胞,E-mail:634800634@qq.com
基金资助:
内蒙古自然科学基金重大资助项目(2012zd03)

Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene

LIU Huai-yu^1,2, LI Hao-tian^1,2, SU Xiao-hu^1,2, MENG Fan-hua^1,2, LIU Chun-xia^1,2, ZHANG Yan-ru^1,2, CAO Jun-wei^1,2

1. College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Key Laboratory of Inner Mongolia Autonomous Ragion Biological Manufacturing, Hohhot 010018, China

Received:2015-06-23 Online:2016-01-20 Published:2016-01-23

摘要/Abstract

摘要： Sox2是多能干细胞的主要标记之一,有研究发现高表达Sox2基因的神经干细胞作为供体细胞进行核移植时具有较高的重编程能力。本研究旨在通过对绵羊骨髓间充质干细胞(bonemarrowmesenchymal stem cells,BMSC) Sox2基因进行外源性增强表达,以期提高其重编程能力,从而改善动物体细胞克隆效率。试验提取绵羊胎儿生殖腺组织RNA,以其为模板克隆Sox2基因cDNA序列,装入真核表达载体pEGFP-N1,构建出pEGFP-N1-Sox2表达载体。经脂质体转染将重组质粒转染入绵羊BMSC,经G418与荧光标记双筛选后挑选单克隆并扩增培养。测序鉴定表明,克隆得到绵羊Sox2基因CDS区全长,重组质粒构建成功;荧光检测表明,成功建立表达Sox2基因的绵羊BMSC系。本研究得到了高表达Sox2基因的绵羊BMSC系,为提高体细胞克隆过程中的重编程效率提供了新思路。

关键词: Sox2基因; 骨髓间充质干细胞; 重编程

Abstract: Sox2 is one important marker of pluripotent stem cells, a study found that neural stem cells with high expression of Sox2 as donor cells showed higher reprogramming ability in nuclear transplantation.In this study, through enhancing exogenous Sox 2 gene expression of sheep bone marrow mesenchymal stem cells, in order to raise their reprogramming ability, and improve the efficiency of somatic cell cloning in animal.Total RNA was extracted from sheep testicular tissue, and with this template, Sox2 cDNA sequence was amplified and inserted into the eukaryotic expression vector pEGFP-N1 to build a recombinant vector pEGFP-N1-Sox2.The vector was transfected into the sheep bone marrow mesenchymal stem cells by liposome method, and through G418 and fluorescence screening to obtain and amplify monoclone.DNA sequencing showed that sheep Sox 2 gene CDS sequence was obtained, and recombinant plasmid was successfully constructed.Identification of fluorescence confirmed that stable sheep bone marrow mesenchymal stem cell lines transfected with Sox2 were established.This study obtained the sheep bone marrow mesenchymal stem cell lines with high expression of Sox2, and provided a new idea for raising reprogramming efficiency in the process of somatic cell cloning.

Key words: Sox2 gene; bone marrow mesenchymal stem cells; reprogramming

中图分类号:

Q813

刘怀禹, 李浩天, 苏小虎, 孟凡华, 刘春霞, 张焱如, 曹俊伟. Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立[J]. , 2016, 43(1): 16-22.

LIU Huai-yu, LI Hao-tian, SU Xiao-hu, MENG Fan-hua, LIU Chun-xia, ZHANG Yan-ru, CAO Jun-wei. Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene[J]. , 2016, 43(1): 16-22.

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Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立

Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene

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