title: Downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells.
abstract: RNA interference (RNAi) is a recently observed process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA (mRNA) in animal and plant cells. In several model systems, RNAi had been developed into a useful tool for the investigation of gene function. In order to study the effectiveness of RNAi in mammalian cells, we introduced chemically synthetic 21-nucleotide small interference RNA (siRNA) duplexes into 293T/GFP cells, which were transduced by enhanced green fluorescence protein (EGFP) gene, by means of TransIT-TKO, Oligofectamine reagent, Lipofectamine 2000 respectively. The results demonstrated that EGFP expression was significantly and specifically inhibited by the corresponding dsRNA, but not by unrelated dsRNA. In three different vectors, Lipofectamine 2000 demonstrated the highest transfection efficiency with a 48 h exposure. The decrease in EGFP fluorescence intensity was approximately 80%. Although TransIT-TKO and Oligofectamine displayed similar trends, the transfections were inefficient, and often toxic. The results also exhibited that siRNA inhibited the EGFP gene expression in a dose and time-dependent manner. Therefore, we concluded that the Lipofectamine 2000 was a better transfection reagent for RNAi. RNAi pathway seems operative in mammalian embryo cells. RNAi may be developed into a potential tool for gene therapy.
authors.affiliation: Department of Pulmonary Diseases, Zhongshan Hospital, Fudan University, Shanghai, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.forename: Chun-Xue
authors.initials: CX
authors.lastname: Bai
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Ling
authors.initials: L
authors.lastname: Mao
authors.forename: Lei
authors.initials: L
authors.lastname: Gao
mesh_terms: D002460:Cell Line; D015536:Down-Regulation; D020868:Gene Silencing; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000295:instrumentation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006801:Humans; D007372:Interferons / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007372:Interferons