Screening of siRNA target sequences by using fragmentized DNA.
BACKGROUND
RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms. In mammals, RNAi can be induced by using short interfering RNA (siRNA). The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences.


METHODS
We developed an siRNA target sequence selection system by first constructing parallel-type siRNA expression vector libraries carrying siRNA expression fragments originating from fragmentized target genes, and then using a group selection system. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes.


RESULTS
We carried out the first screening of groups containing more than 100 random siRNA expression plasmids in total for each target gene, and successfully obtained target sequences with very strong efficacy. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity.


CONCLUSIONS
This system should allow us to perform screening for powerful target sequences, by including all possible target sequences for any gene, even without knowing the whole sequence of the target gene in advance. At the same time, target sequences that should be avoided due to cytotoxicity can be identified.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
Vivi
V
Kasim
Kazunari
K
Taira
Makoto
M
Miyagishi
D001483:Base Sequence; D015152:Blotting, Northern; D016923:Cell Death; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D013379:Substrate Specificity
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004247:DNA