LRIG3特异性RNA干扰真核表达载体的构建及稳定株筛选  

Construction of LRIG3 specific short-hairpin RNA expressing vector and screening of stably transfected cell clone

作　　者：蔡明俊[1] 谢蕊繁[1] 韩林[1] 陈如东[1] 王宝峰[1] 叶飞[1] 郭东生[1] 雷霆[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经外科,湖北武汉430022

出　　处：《中华神经外科疾病研究杂志》2009年第2期122-126,共5页Chinese Journal of Neurosurgical Disease Research

基　　金：国家自然科学基金资助项目(30500521)

摘　　要：目的构建LRIG3(leucine-rich repeats and immunoglobulin-like domains 3,LRIG3)基因特异的RNA干扰质粒,为探讨抑制LRIG3基因表达对脑胶质瘤细胞生物学行为调控的研究奠定基础。方法根据GenBank数据库提供的LRIG3基因核苷酸序列,选择设计2条能转录短发卡RNA(shRNA)的DNA序列,命名为LRIG3-shRNA1、LRIG3-shRNA2,同时设计1条非特异性序列作为阴性对照,命名为negative-shRNA。并与pGenesil2质粒载体连接,转化感受态大肠杆菌,挑选阳性克隆,抽取重组质粒,使用限制性内切酶SalI酶切电泳,DNA测序鉴定。3种重组表达载体转染胶质瘤细胞系GL15细胞,用G418筛选后挑选单克隆并扩增获得稳定株。逆转录酶-聚合酶链反应(RT-PCR)和Western blot分别在mRNA和蛋白水平上检测LRIG3的表达。结果重组质粒成功转化感受态大肠杆菌,经SalI酶切琼脂糖凝胶电泳分析,结果表明寡核苷酸成功插入到预计位点,经测序鉴定,序列完全正确。G418筛选出稳定转染三种质粒的GL15细胞,转染pGenesil2-LRIG3-shRNA组细胞LRIG3mRNA和蛋白表达明显低于转染pGenesil2-negative-shRNA组。结论成功构建针对LRIG3基因的特异性shRNA真核表达载体,转染细胞后可抑制LRIG3基因表达,为进一步研究其基因功能奠定了基础。Objective To construct eukaryotic expression vectors of RNA interference specific for LRIG3 gene, and to screen the stably transfected cell clone. Methods Genomic sequences of LRIG3 gene was retrieved from Genbank and cDNA was designed encoding shRNA （ small hairpin RNAs ） for LRIG3. The cDNA was synthesized and inserted into plasmid pGenesil2. Recombinant vectors were then transformed into competent E. coli. The positive clones were selected and recombinant plasmids were extracted. The plasmids were digested with Sal Ⅰ and loaded in agarose gel electrophoresis. The three shRNA vectors were transfected into GL15 by Metafectene. The stably transfected cell clones were obtained after being screened with G418. Reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting were performed to examine the inhibitory effect at the RNA level and protein level. Results The stably transfected pGenesil2-LRIG3-shRNA cell clones was significantly down-regulated by siRNA as validated by RT-PCR and Western blotting. Conclusion RNA interfering （RNAi） mediated by the shRNA expression vector can significantly down-regulate the expression of LRIG3 in glioma cell line GL15. The stable transfected cell clone is obtained for further study.

关 键 词：LRIG3基因 RNA干扰 脑胶质瘤细胞 载体构建 

分 类 号：R739.41[医药卫生—肿瘤]