沉默RANK真核表达载体的构建及有效干扰序列的筛选

目的构建沉默小鼠RANK基因的真核表达载体pSuper-shRANK,筛选沉默RANK的最佳干扰序列。方法针对小鼠RANK基因设计并合成3对干扰序列,将其连接到真核表达载体pSuper上,酶切及测序鉴定正确后命名为pSuper-shRANK,后将其用脂质体瞬时转染小鼠骨髓巨噬细胞,用荧光显微镜观察转染情况,用Real-timePCR和Westernblot分析干扰效率。结果酶切分析与测序结果表明重组载体pSuper-shRANK构建成功,脂质体共转染后,Real-timePCR和Western blot分析结果显示shRANK-3干扰效果最好,干扰效率达88.7%,筛选出shRANK-3为最佳干扰序列。结论成功构建了沉默小鼠RANK基因的真核表达载体pSuper-shRANK;筛选出了沉默小鼠RANK的最佳干扰序列,为研究抑制该基因在破骨细胞分化及活化作用机制中奠定了基础。

Construction of eukaryotic expression vector with shRNA targeting RANK and screening for optimal shRNA

Objective To construct an eukaryotic expression vector pSuper-shRANK with short hairpin RNA （shRNA） targeting mouse RANK gene,and to screen an optimal shRNA targeting RANK.Methods Three pairs of shRNAs targeting RANK were designed and synthesized,which were cloned into pSuper.After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pSuper-shRANK,which was subsequently transfected into mouse bone marrow-derived macrophages （BMM） using Lipofectamine 2000.The optimal shRNA was selected among three pairs of shRNAs by RANK expression analysed by Real-time PCR and Western blot.Results Both restriction enzyme digestion and sequencing assays showed that the recombinant vector pSuper-shRANK was successfully constructed.The results of Real-time PCR and Western blot showed that shRANK-3 produced significant reduction （88.3%） on RANK expression （P〈0.01） among the three shRNAs.ConclusionsThe eukaryotic expression vector pSuper-retro-puro with shRNA targeting mouse RANK gene was successfully constructed and the optimal shRNA targeting RANK was also identified.This study has paved a way for future research on biological functions of mouse RANK gene,in particular its role in osteoclastogenesis of mouse BMMs.