靶向大鼠gnas基因shRNA真核表达载体的构建与鉴定

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Gs a 蛋白在心力衰竭中作用的体内外实验奠定了基础.

Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to gnas gene

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 a).Methods: Three pairs of shRNAs that target at gnas gene were designed. After annealed,the inserts were ligated into the linearized pGenesil-1.1 plasmids. The eukaryotic expression plasmids were constructed and identified using restriction enzyme analysis and sequencing analysis. The GAPDH plasmid was constructed as a positive control and targeting no-isogeny gene was served as a negative control.Results:pGenesil-1.1 plasmids were confirmed by restriction enzyme analysis and sequencing analysis in accordance with design requiring. Conclusion: We successfully constructed 3 recombinant plasmids pGenesil-1.1-shRNA targeted to gnas gene,and it's helpful for further research the role of Gs a gene in heart failure by RNA interference technique.