摘要
目的:利用PCR构建RNA干扰质粒载体的方法,促进RNA干扰技术的广泛开展。方法:PCR方法获得小鼠U6+27启动子序列,以及干扰绿色荧光蛋白(EGFP)表达的siRNA的相应DNA模板序列。将其序列克隆入pUC19,获得RNA干扰载体pU6-siEGFP。将pU6-siEGFP及pcDNA3.1/EGFP共转导COS-7细胞,观察EGFP表达情况。结果:所构建的pU6-siEGFP能够成功干扰COS-7细胞中EGFP的表达。结论:PCR方法成功构建了RNA干扰载体,为RNA干扰技术在更多实验室的广泛开展奠定基础。
Objective:To facilitate the use of RNA interference technique in laboratory research, we here made a detailed description of the construction of a siRNA expressing plasmid by PCR method. Methods:The U6+27 promoter cassette and siEGFP sequence were obtained by PCR method. They were cloned into pUC19. The resultant plasmid pU6-siEGFP was co-transfected into COS-7 with pcDNA3.1/EGFP. Green fluorescent expression was observed under fluorescent microscopy. Results:The plasmid pU6-siEGFP can efficiently reduce the expression of EGFP in COS-7 cells. Conclusion:We successfully constructed the siRNA expressing plasmid by PCR method. The construction process is applicable in many laboratories, which facilitates the wider use of RNA interference technique.
出处
《南京医科大学学报(自然科学版)》
CAS
CSCD
北大核心
2005年第6期389-391,F003,共4页
Journal of Nanjing Medical University(Natural Sciences)
基金
西安交通大学第二医院科研基金资助项目(2001YJ-07)
关键词
RNA干扰
绿色荧光蛋白
多聚酶链反应
RNA interference
enhanced green fluorescent protein
polymerase chain reaction