靶向大鼠gnas基因shRNA真核表达载体的构建与鉴定

作者:田运娇;易岂建;米青;李娅莎 刊名:重庆医科大学学报 上传者:刘亚军

【摘要】目的:构建并鉴定靶向大鼠gnas基因的小干扰RNA(short hairpin RNA,shRNA)真核表达载体。方法:根据大鼠gnas基因mRNA序列设计并合成3条shRNA特异性寡核苷酸片段,退火形成双链后克隆进入线性化pGenesil-1.1载体,并进行酶切鉴定和测序,同时构建针对大鼠GAPDH基因的阳性对照质粒和不具有基因同源性的非特异性基因的质粒做阴性对照。结果:经酶切和测序鉴定分析,构建的shRNA已成功插入载体,并且与设计序列完全相符。结论:成功构建了靶向大鼠gnas基因的shRNA真核表达载体,为后续研究Gsα蛋白在心力衰竭中作用的体内外实验奠定了基础。

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一10一 重庆医科大学学报2010年第35卷第1期(Journal of Chongqing Medical Univem耐2010.V01.35 No.1) 基础研究文章编号:0253—3626(2010)01-0010--04 靶向大鼠gnas基因shRNA真核表达载体的构建与鉴定 田运娇-,易岂建-,米青-,李娅莎2 (1.重庆医科大学附属儿童医院心脏中心;2.重庆医科大学儿科研究所中心实验室,重庆400014) 【摘要1目的:构建并鉴定靶H大鼠gnas基因的小干扰RNA(shorthairpinRNA,shRNA)真核表达载体。方法:根据大鼠gnas基因mRNA序列设计并合成3条shRNA特异性寡核苷酸片段,退火形成双链后克隆进入线性化pGenesil一1.1载体,并进行酶切鉴定和测序,同时构建针对大鼠GAPDH基因的阳性对照质粒和不具有基因同源性的非特异性基因的质粒做阴性对照。结果:经酶切和测序鉴定分析,构建的shRNA已成功插入载体,并且与设计序列完全相符。结论:成功构建了靶向大鼠gnas基因的shRNA真核表达载体,为后续研究Gs 0【蛋白在心力衰竭中作用的体内外实验奠定了基础。 【关键词】gnas基因;RNA干扰;质粒;心力衰竭 【中国图书分类法分类号】R318.11 【文献标识码】A 【收稿日期】2009-09-07 Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to gnas gene 一 一 一 一 TIAN Yun-jiao,et al (Heon Center,the Children’s Hospital,ChongqingMedical University) 【Abstr∽.t]Objective:To construct eukaryotic expression plasmids containing short hairpin RNA(shRNA)that target at rat gnas gene,which encodes the a--subunit of the Gs protein(Gs d).Methods:Three pairs of shRNAs that target at gnas gene we陀designed.After annealed,the inserts were ligated into the linearized pGenesil一1.1 plasmids.The eukaryotie expression plasmids were constructed and identified using restriction enzyme analysis and sequencing analysis.The GAPDH plasmid wa8 constructed as a positive control and targeting no—isogeny gene was served as a negative contr01.Results:pGenesil一1.1 plasmids were confirmed by restriction enzyme analysis and sequencing analysis in accordance with design requiring.C

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