小鼠IGFBP-6基因shRNA的设计与筛选

Construction and screening of the shRNA for the IGFBP-6 gene in mouse

胰岛素样生长因子结合蛋白6(IGFBP-6)是胰岛素样生长因子结合蛋白家族(IGFBPs)的一员,IGFBP-6不同于IGFBP1-5,其与IGF-2的亲和力显著的优先于IGF-1,受到研究者的广泛关注。本研究在小鼠IGFBP-6基因mR-NA的806、760和908bp处找到潜在靶位点,设计3条shRNA干扰载体,并合成DNA Oligo,体外退火成双链DNA,插入pGU6/GFP/Neo载体,获得3条针对目的基因不同靶序列的干扰载体,瞬时转染NIH-3T3小鼠胚胎成纤维细胞48h后,利用流式细胞技术分选收集转染重组载体的细胞,提取细胞总RNA和蛋白质,实时荧光定量PCR和酶联免疫吸附法分别检测目的基因mRNA水平,蛋白水平对IGFBP-6的表达。结果表明,IGFBP-6(IGFBP-6,806-826)表现出了最高的干扰效率,转染效率为(79.2±2.3)%时,mRNA水平的沉默效率达(68.9±12.2)%,(P<0.05);蛋白质水平的沉默效率达(46.7±11.3)%,(P<0.05)。本试验为研究IGFBP-6基因对细胞增殖的作用和体内生物学功能奠定了基础。

Insulin-like growth factor binding protein 6 （IGFBP-6） is a member of insulin-like growth factor binding protein family （IGFBPs）. IGFBP-6 is different from IGFBP1-5 which was given extensive attention by researchers because it has high affinity with IGF-2 compared with IGF-1. In this research, three potential sites in mouse IGFBP-6 mRNA sequence （start from 806, 760,908 bp,seperately） were selected. Oligo DNAs of target sequences were synthesized and annealed to form a double-stranded DNA which will be inserted into pGU6/GFP/Neo vector. Three interference vectors for IGFBP-6 were obtained. The three reconstructed vectors and one negative vector were transfected into NIH-3T3 cells separately. After 48 h, cells were sorted and collected by flow cytometry,then the total RNA and protein were extracted. The mRNA expression levels of IGFBP-6 were tested with Real-time PCR and the concentrations of protein were detected by ELISA. The result showed that group of IGFBP-6（IGFBP-6,806-826） had the highest interfering efficiency [IGFBP-6 mRNA reduced（68.9±12.2）%, P〈0. 05 and IGFBP-6 protein reduced（46.7± 11.3） % ,P〈0.05 when the transfection efficiency was（79.2± 2.3）%, P〈0. 053. In this study, we obtained the effective shRNA interference vectors which would lay the foundation for further in vivo studies of the biological function of IGFBP-6 gene.