慢病毒表达载体的构建及沉默FOXQ1基因在大肠癌细胞系DLD-1中的表达  

Construction of lentiviral expression vectors to silenceexpression of FOXQ1 gene in colorectal cancer cell line DLD-1

作　　者：白璇[1] 唐慧[2] 郎丰超[3] 郭强[1] 

机构地区：[1]昆明医科大学 云南省第一人民医院(昆明医科大学附属昆华医院)消化内科,云南省昆明市650032 [2]云南省第一人民医院(昆明医科大学附属昆华医院)临床基础医学研究所,云南省昆明市650032 [3]中国科学院昆明动物研究所,云南省昆明市650032

出　　处：《世界华人消化杂志》2014年第19期2752-2757,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.81260323;云南省科技厅-昆明医科大学联合基金资助项目;No.2012FB095;昆明医科大学研究生创新基金资助项目;No.2012J004;云南省中青年学术技术带头人后备人才培养基金资助项目;No.2013HB083~~

摘　　要：目的:构建FOXQ1基因shRNA慢病毒干扰系统,沉默FOXQ1基因在大肠癌细胞系DLD-1中的表达.方法:根据FOXQ1基因的序列,设计合成3对shRNA干扰序列,退火后连接到载体质粒PLKO.1-puro,将重组质粒转化至STBl3感受态中,涂板培养挑取单菌落,摇菌后小提质粒,选取酶切及测序鉴定正确的重组质粒去内毒素大提,将质粒与辅助包装质粒pRSV-rev、pMDlg-pRRE和pCMV-VSV-G利用磷酸钙法共转染293-T细胞,收集病毒上清,感染目的细胞DLD-1,嘌呤霉素筛选FOXQ1沉默细胞,经荧光定量PCR及Western blot检测干扰效果.结果:酶切及测序结果显示shRNA成功插入载体PLKO.1-puro中,共转染293-T细胞成功获取病毒上清,感染DLD1细胞,经嘌呤霉素筛选出FOXQ1沉默细胞,荧光定量PCR及Western blot鉴定FOXQ1基因表达最高抑制率为90.4%.结论:通过构建FOXQ1基因shRNA慢病毒干扰系统,成功获取FOXQ1基因沉默细胞.为后续FOXQ1基因在肿瘤发生发展的研究奠定实验基础.AIM： To construct lentiviral vectors to silence the expression of the FOXQ1 gene in colorectal cancer cell line DLD-1.METHODS： According to the FOXQ1 gene se-quence, three pairs of shRNAs were designed and synthesized, and then ligated to the lentivi-ral PLKO.1 vector. After the resulting plasmids were transfected into STB13 competent cells and cultured on ampicillin resistant culture plates, single colonies were picked to prepare plasmid DNA. The plasmids were identified by enzyme digestion and DNA sequencing. The identified plasmids and auxiliary packaging plasmids including pRSV-rev, pMDlg-pRRE, pCMV-VSV-G were jointly infected into 293-T cells by the calcium phosphate transfection method, and the lentivirus supernatants were obtained and used for infecting DLD-1 cells. After selection with puromycin, the FOXQ1-silenced cells were obtained. The efficiency of gene knockdown was then determined by real-time PCR and Western blot.RESULTS： Enzyme digestion and DNA se-quencing results showed that the shRNAs were successfully inserted into the PLKO.1 vector. Re-combinant plasmids were transfected into 293-T cells and high-titer lentiviruses were formed. The lentiviruses was transduced into DLD-1 cells. FOXQ1-silenced cells were obtained after selection. Real-time PCR data and Western blot showed that the highest inhibitory efficiency for FOXQ1 expression was approximately 90.4%.CONCLUSION： FOXQ1-silenced cells have been successfully obtained by constructing a lentivirus interference system, which provide an experimental foundation for further study of the function of the FOXQ1 gene in tumorigenesis.

关 键 词：FOXQ1基因 慢病毒载体 RNA干扰 基因沉默 

分 类 号：R735.34[医药卫生—肿瘤]