摘要
通过GenBank数据库检索,基于小鼠MMP-9基因设计特异性的siRNA干扰靶点,克隆至pGCsi-U6/Neo/GFP载体中,使用PEI衍生物包裹,转染小鼠黑色素瘤B16细胞,流式细胞仪和激光扫描共聚焦显微镜分析细胞转染情况,RT-PCR检测24,48 h后MMP-9基因转录水平变化,筛选最佳的重组质粒和干扰靶点。结果显示:成功设计和构建了3个MMP-9-siRNA干扰质粒,3个重组质粒对B16细胞的转染率分别为60.04%、63.93%和56.27%,且3个重组质粒均能有效干扰B16细胞MMP-9 mRNA的表达,其中MMP-9-siRNA-2干扰效率最高(63%),可持续干扰MMP-9基因表达。这些结果提示,MMP-9-siRNA-2为沉默小鼠黑色素瘤细胞MMP-9基因最优的siRNA重组质粒。
Specific siRNA interference target sites were designed according to the sequence of mouse MMP-9 gene in GenBank,and oligonucleotides were synthesized and inserted into pGCsi-U6/Neo/GFP plasmid to constitute the recombinant plasmids,the siRNA recombinant plasmids were transfected into B16 cells by PEI derivative,which was confirmed by flow cytometry and laser scanning confocal microscope observation,and the interfering efficiency was evaluated by Real-time quantitative RT-PCR analysis.The results showed that three MMP-9-siRNA interference plasmids were designed and constructed successfully.Transfection efficiency of the three MMP-9-siRNA recombinant plasmids were 60.04%,63.93% and 56.27%,respectively,and the expression of MMP-9 mRNA in B16 cells was effectively down-regulated.The MMP-9-siRNA-2 had the highest interference efficiency（63%） and persistent interference.The results suggested that MMP-9-siRNA-2 is the optimal recombinant plasmid for？silencing MMP-9 gene of？mouse B16？cell.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2012年第9期906-910,共5页
Chinese Journal of Cell Biology
基金
重庆市教委科学技术研究(No.KJ090302)资助项目~~
