DNA甲基转移酶3A siRNA 真核表达载体的构建和鉴定-东南大学学报医学版

DNA甲基转移酶3A siRNA 真核表达载体的构建和鉴定

单位：1.东南大学基础医学院,遗传与发育生物学系,江苏,南京,210009;东南大学,发育与疾病相关基因教育部重点实验室,江苏,南京,210009； 2.江苏省疾病预防控制中心,江苏,南京,210009

分类号：Q782

出版年·卷·期（页码）：2008·27·第一期（1-5）

摘要：

目的:构建DNA甲基转移酶3A(DNA methyltransferases 3A,DNMT3A)siRNA重组质粒稳定表达载体,为进一步探讨DNMT3A在多种生物学过程及肿瘤发生发展中的机制提供工具.方法:依据DNMT3A基因的cDNA序列,利用siDESIGN软件获得RNAi靶位点后设计出对应的siRNA寡核苷酸模板,体外合成寡核苷酸片段经退火形成短双链,克隆到真核表达载体pSUPER-EGFP1中,构建DNMT3A siRNA重组质粒载体,经酶切鉴定后转染人肝癌细胞系SMMC-7721,荧光实时定量PCR初步分析DNMT3A经RNA干涉后的沉默效应.结果:成功构建了靶向DNMT3A基因的siRNA重组稳定表达载体,此载体有效地降低了肝癌细胞系中DNMT3A基因的表达水平.结论:通过该方法初步确定了沉默DNMT3A基因的较佳靶点.

Objective To investigate the function of DNA methyltransferases 3A(DNMT3A)in a series of biological process and carcinogenesis and to construct an eukaryotic siRNA targeting on DNMT3A to suppress the expression of DNMT3A gene.Methods Using siDESIGN software,the candidate oligonucleotide sequences were designed upon the DNMT3A cDNA.After annealing,the formed double-stranded DNA was ligated with pSUPER-EGFP1 vector.The recombinant was identified by electrophoresis,and then introduced into SMMC-7721 mediated by Lipofectamine 2000.siRNA-induced silencing of DNMT3A gene was determined by using real-time reverse transcriptase PCR at RNA level.Results Vector-derived siRNAs specifically targeting the coding region of DNMT3A gene were successfully constructed,which effectively knocked down DNMT3A in hepatocellular carcinoma cell line SMMC-7721.ConclusionBy RNAi technique,the more effective target silence sites of DNMT3A have been screened from candidates,which may be a practical tool for further research of DNMT3A function.

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