Quantitative and temporal analysis of gene silencing in tumor cells induced by small interfering RNA or short hairpin RNA expressed from plasmid vectors.
Vector-based RNA interference (RNAi) has attracted great interest, because of its more prolonged gene silencing effect compared with small interfering RNA (siRNA). However, the intensity and duration of vector-based RNAi effect has received little attention. In this study, the gene silencing kinetics of short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) driven by U6, H1 or tRNA promoter (pU6-shLuc, pH1-shLuc, and ptRNA-shLuc) was studied in melanoma cells expressing firefly luciferase. A bootstrap method-based moment analysis was performed to statistically and quantitatively evaluate the profile of gene silencing. The analysis showed that pU6-shLuc induced a significantly greater and longer gene silencing than that produced by other promoter-driven shRNA expression vectors. In addition, it was found that pU6-shLuc was at least 100-fold more potent in gene silencing than siRNA targeting the same gene on a numerical basis. These statistical considerations demonstrated that U6 promoter-driven shRNA expressing pDNA is the most effective in inducing gene silencing effect as far as the intensity and duration of RNAi effect is concerned.
Takahashi
Yuki
Y
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Yamaoka
Kiyoshi
K
Nishikawa
Makiya
M
Takakura
Yoshinobu
Y
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D020868:Gene Silencing / Q000502:physiology*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D049409:Luciferases, Firefly / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D008546:Melanoma, Experimental; D051379:Mice; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000737:chemistry; D013997:Time Factors
D004279:DNA, Viral; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly