Expression strategies for short hairpin RNA interference triggers

Abstract

Since the discovery that the triggers for RNA interference (RNAi), small interfering RNAs, could mediate silencing in mammalian cells without triggering a toxic response, RNAi has become the standard tool for sequence-specific knockdown of gene expression in molecular biology. This is due in part to the development of methods for promoter-based expression of RNAi triggers that can mediate stable silencing in mammalian cells. Numerous systems with slightly different characteristics exist, but despite incredible progress in a field that moves very rapidly, challenges still remain. The biggest challenge is to successfully and safely apply RNAi in vivo. Aside from potential issues of delivery, which is one of the most important considerations, successful application of short hairpin RNAs (shRNAs) in vivo requires expression systems that yield potent and specific knockdown of the target in the absence of toxicity. With a couple of exceptions, the current systems available for shRNA expression have not generally resulted in unexpected toxicities, while still providing strong knockdown of the intended targets; however, we do not know enough about how sequence-specific off-target effects will affect various cell and tissue types, or to what extent ectopic expression of RNAi triggers will perturb the endogenous RNAi mechanisms.

FIG. 1

Expression strategies for shRNAs in mammalian cells. (A) The Pol III promoters based on U6 and H1 directly transcribe the shRNAs from the +1 position of the promoter transcription units. Termination occurs within a stretch of several uracils in the transcript. The shRNAs contain 5′-triphosphates (Paul et al., 2002). (B) U1 snRNA transcription unit for shRNA. The transcripts initiate at +1 and the first nucleotide is capped. U1 has a terminator element that is positioned immediately after the shRNA. The terminator is processed from the transcript (Denti et al., 2004). (C) tRNA Pol III transcription unit for shRNA. The shRNA is appended immediately after the stem of the tRNA following the discriminator nucleotide (Scherer et al., 2007). The shRNA is processed from the tRNA leaving a 5′-monophosphate. The tRNA transcription units have A- and B-box regulatory elements and terminate in a string of uracils. (D) Generalized Pol II transcription system for microRNA mimics. The promoter element can contain tissue-specific enhancers and a TATA element. The transcripts will initiate with a cap structure and a leader element. The microRNA mimic is processed from the primary transcript by Drosha/DGCR8, resulting in a pre-miRNA mimic (Zeng et al., 2002).

FIG. 1

Expression strategies for shRNAs in mammalian cells. (A) The Pol III promoters based on U6 and H1 directly transcribe the shRNAs from the +1 position of the promoter transcription units. Termination occurs within a stretch of several uracils in the transcript. The shRNAs contain 5′-triphosphates (Paul et al., 2002). (B) U1 snRNA transcription unit for shRNA. The transcripts initiate at +1 and the first nucleotide is capped. U1 has a terminator element that is positioned immediately after the shRNA. The terminator is processed from the transcript (Denti et al., 2004). (C) tRNA Pol III transcription unit for shRNA. The shRNA is appended immediately after the stem of the tRNA following the discriminator nucleotide (Scherer et al., 2007). The shRNA is processed from the tRNA leaving a 5′-monophosphate. The tRNA transcription units have A- and B-box regulatory elements and terminate in a string of uracils. (D) Generalized Pol II transcription system for microRNA mimics. The promoter element can contain tissue-specific enhancers and a TATA element. The transcripts will initiate with a cap structure and a leader element. The microRNA mimic is processed from the primary transcript by Drosha/DGCR8, resulting in a pre-miRNA mimic (Zeng et al., 2002).