Construction and identification of vectors expressing short hairpin RNA targeting mouse STAT4 and STAT6 gene

YANG Qin; et al

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Construction and identification of vectors expressing short hairpin RNA targeting mouse STAT4 and STAT6 gene

更新时间：2024-01-17

- Construction and identification of vectors expressing short hairpin RNA targeting mouse STAT4 and STAT6 gene
- Journal of Chongqing Medical University Vol. 34, Issue 1, Pages: 16-19(2009)
- 作者机构：
  
  重庆医科大学附属儿童医院呼吸科
- 作者简介：
- 基金信息：
- DOI：
  CLC： R346
- Published：2009，
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[1]杨琴,符州,田代印,刘恩梅,王莉佳,黄英.小鼠STAT4,STAT6基因shRNA真核表达质粒的构建及鉴定[J].重庆医科大学学报,2009,34(01):16-19.

YANG Qin, et al. Construction and identification of vectors expressing short hairpin RNA targeting mouse STAT4 and STAT6 gene. [J]. Journal of Chongqing Medical University 34(1):16-19(2009)
[1]杨琴,符州,田代印,刘恩梅,王莉佳,黄英.小鼠STAT4,STAT6基因shRNA真核表达质粒的构建及鉴定[J].重庆医科大学学报,2009,34(01):16-19. DOI：

YANG Qin, et al. Construction and identification of vectors expressing short hairpin RNA targeting mouse STAT4 and STAT6 gene. [J]. Journal of Chongqing Medical University 34(1):16-19(2009) DOI：

摘要

目的:构建并鉴定小鼠信号转导子和转录激活因子4（STAT4）

信号转导子和转录激活因子6（STAT6）具有发夹结构小干扰RNA（shRNA）表达质粒。方法:根据STAT4和STAT6基因mRNA序列

在体外分别合成有小发夹结构的2条STAT4特异性寡核苷酸序列

2条STAT6特异性寡核苷酸序列

退火后与线性化pGenesil-3质粒连接

转化感受态细胞DH5α

扩增

纯化得到所需质粒。同时设计构建分别针对小鼠甘油醛-3-磷酸脱氢酶（GAPDH）的干扰质粒作为阳性对照

不具有基因同源性的非特异性基因的质粒作为阴性对照序列。通过酶切后琼脂糖凝胶电泳和基因测鉴定纯化质粒分子量及插入片段的序列。结果:经限制性酶切和测序鉴定分析证实基因插入正确

与设计的序列完全相符。结论:成功构建了针对小鼠STAT4

STAT6基因的发夹结构小干扰RNA表达载体。为今后利用基因沉默技术从转录后水平抑制STAT4

STAT6的研究奠定基础。

Abstract

Objective: To construct short-hairpin RNA(shRNA)expressing vectors aimed at mouse signal transducers and activators of transcription-4/6(STAT4/STAT6). Methods: Target sequences in the middle of STAT4 and STAT6 gene were selected

and then the complementary 65 mer oligonucleotides of each sequence were synthesized. After annealed

the inserts were ligated into the linearized pGenesil-3 plasmid. The plasmids were transformed into DH5α bacteria to amplify and then purified. GAPDH plasmid was constructed as a positive control and targeting none gene was served as a negative control. These purified plasmids were identified by gel electrophoresis after being cut with restriction enzyme and sequencing. Results: The results of gel electrophoresis and sequencing showed that the plasmids were identical with the positive control

and the sequences were identical with what we have inserted in. Conclusion: The vectors which can express siRNA hairpin aimed at STAT4/STAT6 gene have been constructed successfully and it lays the groundwork for the further study.

关键词

哮喘信号传导子及转录激活因子RNA干扰shRNA质粒

Keywords

asthmaSignal transducers and activators of TranscriptionRNAishRNAplasmid

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田代印

杨琴

王莉佳

Zhang Xinyue

Shao Zijian

Lian Kang

Related Institution

Department of Respiratory Diseases,the Children’s Hospital,Chongqing Medical University

Department of Respiratory Medicine，Children’s Hospital，Zhejiang University School of Medicine/ National Clinical Research Center for Child Health

Department of Pediatrics，The Affiliated Hospital of Nanjing University of Chinese Medicine，The First Clinical Medical College of Nanjing University of Chinese Medicine

Department of Traditional Chinese Medicine，Children’s Hospital，Zhejiang University School of Medicine/ National Clinical Research Center for Child Health

Department of Emergency and Critical Care Medicine,The First Affiliated Hospital of Chongqing Medical University

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