RNAi Knockdown of Gene Expression

Knockdown of gene expression is accomplished by designing siRNA sequences that target the coding and noncoding regions of the candidate mRNAs with perfect complementarity. In mammalian cells, administration of siRNAs is effective in short-term investigations. Introduction of siRNA into cells is accomplished by transfection or electroporation of either the specific siRNA itself or small hairpin RNA (shRNA) in which the hair loop is rapidly cleaved to produce siRNA. Most of the proposed applications of RNAi incorporate 21-nt siRNA duplexes with 2-nt 3' overhangs, which after chemical synthesis allow large-scale and uniform production of siRNA molecules that are also amenable to stabilizing chemical modifications. Several commercial entities involved in the manufacturing of siRNAs provide effective design algorithms online, which are based on a combination of mRNA target sequence and secondary structures. In some siRNA-target combinations, the use of longer dsRNAs can increase the potency of RNAi (Kim et al. 2005; Siolas et al. 2005).

For stable longer-term suppression, a gene construct coding for the shRNA with a Pol III or a Pol II promoter can be applied (Brummelkamp et al. 2002; Y. Shi 2003; Yu et al. 2002a). RNA Pol II and III promoters are used to drive expression of shRNA constructs (Amarzguioui et al. 2005), depending on the type of expression required. Pol III promoters drive high levels of constitutive shRNA expression, and their transcription initiation points and termination signals are well defined. Pol II promoter-driven shRNAs can be expressed tissue-specifically (Zeng et al. 2002). Expressed shRNAs are efficiently incorporated into RISC, leading to a more potent inhibition of target gene expression. Generally, viral vectors such as adenovirus, lentivirus, or adeno-associated virus can carry the inhibitory construct into cells to achieve adequate expression for knockdown (Kim and Rossi 2007).

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