Cloning of human REG γ cDNA and construction of the stable transfective cell line

TIAN Tian; et al

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Cloning of human REG γ cDNA and construction of the stable transfective cell line

更新时间：2024-01-17

- Cloning of human REG γ cDNA and construction of the stable transfective cell line
- Journal of Chongqing Medical University Vol. 34, Issue 2, Pages: 145-147(2009)
- 作者机构：
  
  重庆医科大学附属第一医院普外科
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q78
- Published：2009，
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[1]田甜,王小毅,李凡,任国胜.人REG γ cDNA克隆及其稳定转染细胞株的建立[J].重庆医科大学学报,2009,34(02):145-147.

TIAN Tian, et al. Cloning of human REG γ cDNA and construction of the stable transfective cell line. [J]. Journal of Chongqing Medical University 34(2):145-147(2009)
[1]田甜,王小毅,李凡,任国胜.人REG γ cDNA克隆及其稳定转染细胞株的建立[J].重庆医科大学学报,2009,34(02):145-147. DOI：

TIAN Tian, et al. Cloning of human REG γ cDNA and construction of the stable transfective cell line. [J]. Journal of Chongqing Medical University 34(2):145-147(2009) DOI：

摘要

目的:构建真核表达重组体PcDNA3.1-REGγ的稳定转染细胞株

为进一步深入研究REGγ的功能奠定基础。方法:提取MCF-7细胞的总RNA为模板

通过RT-PCR获取全长REGγcDNA

经限制性内切酶EcoRⅠ、EcoRⅤ酶切后与PcDNA3.1连接构建重组体

再经内切酶酶切及DNA序列分析鉴定重组体构建正确。以Lipofectamine2000将重组体导入HBL-100细胞

由G418筛选出稳定转染细胞株。结果:经免疫细胞化学和RT-PCR检测并证实稳定转染细胞株中有REGγ的高表达。结论:真核表达重组体PcDNA3.1-REGγ构建成功

并经抗生素筛选获得了稳定高表达REGγ的细胞株

为进一步研究REGγ的功能奠定了基础。

Abstract

Objective:To construct the stable transfective cell line with the eukaryotic expression vector for human REGγcDNA to laid the foundation for further study of the function of REGγ.Methods:REGγcDNA was obtained by RT-PCR of total RNA extracted from the human breast cancer cell MCF-7

and it was digested by the restriction endonuclease EcoRⅠand EcoRⅤbefore connection withPcDNA3.1.The recombination with forward insert were slelected by restriction endonuclease digestion and confirmed correct by sequencing and then transfected into HBL-100 cell by using lipofectamine2000.The stable transfected cell line was selected in medium containing antibiotic(G418).Results:The result of immunocytochemistry and RT-PCR of total RNAextracted from the stable transfected cell line showed that there existed the overexpression of REGγcDNA in it.Conclusion:The construction of the eukaryotic expression vector for REGγwas successful

and the stable transfected cell line for overexpression REGγ was obtained by G418 selection.This work may pave the way for further study of the functions of REGγ.

关键词

REGγcDNA克隆转染

Keywords

REGγcDNA cloneTransfect

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