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224 Cheng and Chang1. IntroductionRNA interference (RNAi) is a mechanism of posttranscriptional gene silencingin which double-stranded RNAs (dsRNAs) induce sequence-specific cleavageof the homologous RNA transcripts and in turn cause complete degradationof the aberrant RNA fragments, resulting in reduced or loss of activities of thegenes (1,2). During the processes of RNAi-mediated gene silencing, the dsRNAsare first recognized and cleaved into 21–23-nucleotide (nt) small interferingRNAs (siRNA) duplexes, with symmetrical 2-nt 3′-overhangs by dsRNA-specificRNase III-related endonuclease, Dicer (3,4). The resulting siRNAs are efficientlyincorporated into the RNA-induced silencing complex (RISC) to form a ribonucleoproteincomplex that first mediates the unwinding of the siRNA duplexes andselectively degrades the sense strand of siRNA. The single-stranded siRNA-coupled(RISC) is in turn guided to catalyze the endonucleolytic cleavage of homologousRNA transcripts at the site where the antisense strand of siRNAs is complementarilybound (5,6). Subsequently, the resulting disruptive RNA fragmentsare immediately subjected to exonucleolytic destruction by the action ofexoribonuclease.RNAi is evolutionarily conserved to each of the eukaryotic organismsinvolved in regulation of the gene activity. The function of RNAi, primarily,appears to be implicated in cellular defense mechanism in antiviral infectionand maintaining genomic integrity against transposable element-inducedgenomic instability (7,8), as well as in cellular gene regulation and chromosomalepigenetic control (9–11). Currently, it has emerged as a practically usedstrategy for reverse functional genomics and in particular as an extremely powerfulapproach for molecular therapeutics (12–15). In plants and invertebrates,introduction of the dsRNAs into the cells induces sequence-specific inhibitionof homologous gene expression. However, in mammals, the dsRNAs longerthan 30 nt in length trigger a strong cytotoxic response through activation of thedsRNA-dependent protein kinase and 2′,5′-oligoadenylate synthetase, resultingin inactivation of the eukaryotic initiation factor-2α and activation of theRNaseL, and in turn causing general inhibition of protein synthesis and nonspecificdegradation of single-stranded RNA, respectively (16–20). However, byusing short synthetic 21-nt siRNAs with symmetrical 2-nt 3′-overhangs allowfor inducing the sequence-specific gene silencing, yet avoid triggering the nonselectivecytotoxic effects by long dsRNAs (21,22).In mammals, there are mainly two strategies in producing dsRNAs by exogenousdelivery of synthetic siRNAs (21,22) or short hairpin RNAs (shRNAs)(23) and endogenous vector-expressed siRNAs (24–26) or shRNAs (27–31).The silencing effect induced by synthetic dsRNAs is transient and the targetgene is reactive after a few days, as well as the cost of chemical synthesis of RNA