摘要
目的:构建小鼠转化生长因子β1(TGF-β1)短发夹RNA(shRNA)真核表达载体,探讨TGF-β1在血管发育中的调控作用。方法:根据GenBank小鼠TGF-β1mRNA序列,设计合成三对短链寡核苷酸,退火后形成双链DNA并克隆至入门载体pEN-mH1c。将插入目的基因片段的入门载体与带有绿色荧光蛋白(GFP)标签的shRNA真核表达载体pDS-hpEy进行LR重组反应,完成三个TGF-β1shRNA表达载体的构建,分别命名为pDS-Ta,pDS-Tb和pDS-Tc。经测序确认后,转染小鼠成纤维细胞(NIH/3T3),筛选稳定表达的细胞克隆,以RT-PCR及Western blot方法检测转染后TGF-β1 mRNA和蛋白表达。结果:RT-PCR和Western blot显示pDS-Tc可明显下调NIH/3T3细胞TGF-β1的mRNA和蛋白表达,mRNA下调约为70%,蛋白表达减少约65%。结论:GFP标签TGF-β1shRNA表达载体能够阻断TGF-β1基因表达,可作为研究TGF-β1调控血管发育机制的一个工具,为阐明TGF-β1信号传导通路奠定基础。
To construct short hairpin RNA（shRNA） eukaryotic expression vectors targeting TGF-β1 for further research on the effects of TGF-β1 on vasculogenesis and angiogenesis. Methods： Three pairs of siRNA target sequences coding from the mRNA of TGF-β1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mHlc entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, tile three resulting TGF-β1 shRNA expression vectors were transfected into the mouse fibroblast cell line （N1H/3T3）, and then cell clones stably expressing TGF-β1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction（RT-PCR） and Westem blot were used to detect the mRNA and protein expression. Results： RT-PCR and Western blot showed that one of the TGF-β1 shRNA expression vectors pDS_Tc downregulated TGF-β1 mRNA and protein expression markedly in NIH/3T3 cells. Conclusion： ShRNA eukaryotic expression vectors targeting TGF-β1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-β1 regu- lates vasculogenesis and angiogenesis.
出处
《中国应用生理学杂志》
CAS
CSCD
北大核心
2009年第2期244-249,共6页
Chinese Journal of Applied Physiology
基金
国家自然科学基金资助项目(30370526)
