南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (04): 483-.

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基于PCR的siRNA表达框的制备

郭秋野； 马文丽； 张宝； 吴清华； 严律； 郑文岭；   

  1. 南方医科大学基因工程研究所； 华南基因中心 广东 广州 510515； 广东 广州 510515； 广东 广州 510830；
  • 出版日期:2006-04-20 发布日期:2006-04-20

Preparation of small interfering RNA expression cassette based on PCR technique

GUO Qiu-ye, MA Wen-li, ZHANG Bao, WU Qing-hua, YAN L(u|¨), ZHENG Wen-ling Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China; Genomic Research Center of South China, Guangzhou 510830, China   

  1. 南方医科大学基因工程研究所； 华南基因中心 广东 广州 510515； 广东 广州 510515； 广东 广州 510830；
  • Online:2006-04-20 Published:2006-04-20

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摘要： 目的改进目前基于PCR的方法制备siRNA表达框,转染细胞后产生高效的RNA干扰作用。方法从K562细胞中提取基因组DNA,以此为模板,PCR扩增U6启动子序列并克隆至pMD18-T载体中,用载体上的引物进行扩增,所得产物即制成作为扩增siRNA表达框的模板。选择p53为靶基因,扩增制备siRNA表达框,测序验证后,转染SH-SY5Y 细胞48h后提取RNA进行RT-PCR,检测RNA干扰的效应。结果测序结果表明所克隆的U6启动子序列正确。细胞转染表达框,在mRNA水平上,p53基因的表达明显受到了抑制。结论本实验所制作的基于PCR的方法制备siRNA表达框可以很好地应用于RNAi作用研究。

Abstract: Objective To modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference. Methods The U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference. Results The sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection. Conclusion The siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research. 

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