miRNA-1真核表达载体的构建及转染骨髓间充质干细胞绿色荧光蛋白的表达

目的 构建大鼠miRNA-1真核表达载体,为研究miRNA-1功能提供实验基础.方法 应用技术体外合成miRNA-1前体对应的基因组片段,定向克隆到pSD11-U6/Neo/绿色荧光蛋白载体上.阳性克隆进行及DNA测序鉴定,将构建成功的重组质粒pSD11-U6/Neo/绿色荧光蛋白/miRNA-1借助脂质体转染大鼠骨髓间充质干细胞,荧光显微镜下观察绿色荧光蛋白表达.结果 PCR鉴定及DNA测序表明真核表达载体pSD11-U6/Neo/绿色荧光蛋白/miRNA-1构建成功.转染大鼠骨髓间充质干细胞后,荧光显微镜下观察到绿色荧光蛋白表达.结论 大鼠miRNA-1真核表达载体构建成功.

Construction of eukaryotic expression vector of miRNA-1 and the expression of green fluorescent protein in transfected bone marrow mesenchymal stem cells

Objective To construct an eukaryotic expression vector for mo-miRNA-1 to provide the experimental basis for miRNA-1 functions.Methods miRNA-1 genomic sequence was amplified by polymerase chain reaction(PCR) and cloned into pSD11-U6/Neo/GFP plasmid.Positive clones were identified with PCR and DNA sequencing, then mesenchymal-like stem cell were transfected pSD11-U6/Neo/GFP/miRNA-1 recombinant plasmid with Lipofectamine.Expression of green fluorescent protein was observed under a fluorescence microscope.Results PCR identification and DNA sequencing showed that the eukaryotic expression vector for miRNA-1 constructed successfully, green fluorescent protein expression was detected after transfection.Conclusion The eukaryotic expression vector for mo-miRNA-1 can be constructed successfully.