Sequences shown in capital letters are fragments originating from the target DNAs. (D) Sequences of iRed2-46**-par, iRed2-50-par and iGFP98-par. Because iRed2-46-par was a mixture of two clones, we assayed each clone and determined that iRed2-46**-par was the one that produced the suppression effect (nearly 80%). (C) Determination of which clone of iRed2-46-par produced the suppressive effect: lane 1, negative control lane 2, iRed2-46-par (mix) lane 3, iRed2-46*-par lane 4, iRed2-46**-par. Compared with the negative control, iGFP98-par produced a 60% suppression effect, whereas others did not produce any obvious suppression. (B) Further assay of each siRNA expression vector against GFP from group 20: lane 1, negative control lane 2, iGFP96-par lane 3, iGFP-97-par lane 4, iGFP98-par lane 5, iGFP99-par lane 6, iGFP100-par. iRed2-46-par and iRed2-50-par produced a nearly 80% suppression effect compared with the negative control, whereas others did not produce any RNAi effect.
(A) Further assay of each siRNA expression vector against DsRed from group 9: lane 1, negative control lane 2, iRed2-46-par lane 3, iRed2-47-par lane 4, iRed2-49-par lane 5, iRed2-50-par. Identifying clones with high RNAi activities. At the same time, target sequences that should be avoided due to cytotoxicity can be identified. 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. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity. 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. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes. 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. The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences. In mammals, RNAi can be induced by using short interfering RNA (siRNA). RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms.
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