Expression of siRNA from a single transcript that includes multiple ribozymes in mammalian cells.
RNA interference (RNAi) has been developed recently as a powerful tool for silencing of mRNAs in various organisms. In mammalian cells, the introduction of small interfering RNAs (siRNAs) can inhibit gene expression in a sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt). Here, we report a method for generating siRNAs in mammalian cells using a self-cleaving ribozyme-expressing vector. Four ribozymes within transcripts that were expressed under the control of a cytomegalovirus (CMV) or tRNAVal promoter excised, in cis, specific sense and antisense sequences from primary transcripts and generated siRNAs in HeLa cells. The siRNAs generated by the ribozymes were able to decrease the expression of a firefly gene for luciferase. These results suggest that polymerase II (pol II) systems, particularly in view of the availability of many potential tissue-specific promoters, and pol III systems, in which siRNAs are generated by a trimming-ribozyme (TRz) system as described here, should be useful in efforts to suppress the expression of specific genes.
Kato
Yoshio
Y
Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan.
Taira
Kazunari
K
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016373:DNA, Antisense / Q000737:chemistry / Q000235:genetics; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008322:Mammals; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D020121:5' Untranslated Regions; D016373:DNA, Antisense; D016337:RNA, Catalytic; D034741:RNA, Small Interfering; D008156:Luciferases