人黑色素浓集激素受体2基因shRNA真核表达载体体外转染

目的构建针对人黑色素浓集激素受体2(MCHR2)的短发夹RNA(shRNA)真核表达载体,并观察其转染效率。方法根据MCHR2基因序列,设计并合成特异性的小干扰片段,将其定向克隆到真核表达载体pGenesil-1中,酶切和测序后,用脂质体转染到HEK293细胞中,用荧光显微镜和流式细胞仪观察荧光表达,鉴定转染效率。结果与结论成功构建4条表达shRNA的质粒及其阴性对照质粒,并成功转染到HEK293细胞系中,转染效率达50%以上。

Transfection of eukaryotic expression vector expressing shRNA section targeting human MCHR2 in vitro

Objective To construct eukaryotic expression vector expressing short hairpin RNA(shRNA) section targeting human MCHR2 and to observe their transfection efficiency. Methods According to the sequence of human MCHR2 gene, the oligonucleotides of shRNA were designed and synthesized and cloned into plasmid pGenesil-1.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vectors were transfected into HEK293 cell line by lipofectamineTM2000,the expression of fluorescence and efficiency of transfection were detected by fluorescence microscopy and flow cytometry.Results Four shRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into HEK293 cells and the transfection efficiency achieved about 50%.