siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells.
RNA interference has emerged as a powerful tool for the silencing of gene expression in animals and plants. It was reported recently that 21 nt synthetic small interfering RNAs (siRNAs) specifically suppressed the expression of endogenous genes in several lines of mammalian cells. However, the efficacy of siRNAs is dependent on the presence of a specific target site within the target mRNA and it remains very difficult to predict the best or most effective target site. In this study, we demonstrate that siRNAs that have been generated in vitro by recombinant human Dicer (re-hDicer) significantly suppress not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras, c-jun and c-fos. In our system, selection of a target site is not necessary in the design of siRNAs. However, it is important to avoid homologous sequences within a target mRNA in a given protein family. Our diced siRNA system should be a powerful tool for the inactivation of genes in mammalian cells.
Kawasaki
Hiroaki
H
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Suyama
Eigo
E
Iyo
Mayu
M
Taira
Kazunari
K
D003001:Cloning, Molecular; D004351:Drug Resistance; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D006367:HeLa Cells; D006801:Humans; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000235:genetics; D011691:Puromycin / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D043244:Ribonuclease III
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D011691:Puromycin; D004722:Endoribonucleases; D043244:Ribonuclease III; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)