Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells.
BACKGROUND
RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells.


METHODS
We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors.


RESULTS
As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five.


CONCLUSIONS
The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.
Miyagishi
Makoto
M
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
Sumimoto
Hidetoshi
H
Miyoshi
Hiroyuki
H
Kawakami
Yutaka
Y
Taira
Kazunari
K
D001483:Base Sequence; D017931:DNA Primers; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology
D017931:DNA Primers; D034741:RNA, Small Interfering; D013752:Tetracycline