miR-133a真核表达载体的构建及转染大鼠心肌H9C2细胞

Construction of miR-133a eukaryotic expression vector and transfection into H9C2 cell

目的:构建miR-133a真核表达载体后,体外转染大鼠H9C2心肌细胞。方法:设计并合成DNA模板引物,用T4DNA连接酶将pre-miR-133a连接到线性化的PgenesIL-1.1质粒中,对重组质粒进行酶切及测序鉴定。以Li-pofectamineTM2000 Reagent将鉴定正确的重组质粒瞬时转染H9C2细胞,用流式细胞仪及倒置荧光显微镜检测转染效率。结果:miR-133a真核表达载体符合设计要求,瞬时转染H9C2细胞的转染率达70%以上。结论:成功构建了miR-133a真核表达载体并转染至大鼠心肌H9C2细胞。

Objective： To construct miR-133a eukaryotic expression vector and transfect it into H9C2 cell.Methods： Design and synthetize the PCR templa-primer.Ligate the pre-miR-133a with linearized PgenesIL-1.1 by T4 DNA Ligase.The recombinants were identified by endonuclease digestion and sequenced.Transient transfect the perfect recombinants into H9C2 cells by LipofectamineTM 2000 Reagent.Finally,detect transfection efficiency by flow cytometer and invert fluorescence microscope.Results： The miR-133a eukaryotic expression vectors were consistent with the design and the transfection efficiency of H9C2 cells was 70%.Conclusion： miR-133a eukaryotic expression vector was successfully constructed and transfected into H9C2 cell.