RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells

Abstract

Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific ␤-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy. RNA interference (RNAi) has become a powerful and widely used tool for the analysis of gene function in invertebrates and plants (reviewed in ref. 1). Introduction of double-stranded RNA (dsRNA) into the cells of these organisms leads to the sequence-specific destruction of endogenous RNAs that match the dsRNA. During RNAi, long dsRNA molecules are processed into 19-to 23-nt RNAs known as short-interfering RNAs (siRNAs) that serve as guides for enzymatic cleavage of complementary RNAs (2-10). In addition, siRNAs can function as primers for an RNA-dependent RNA polymerase that synthesizes additional dsRNA, which in turn is processed into siRNAs, amplifying the effects of the original siRNAs (11,12). Although the overall process of siRNA inhibition has been characterized, the specific enzymes that mediate siRNA function remain to be identified.In mammalian cells, dsRNA is processed into siRNAs (13-16), but RNAi with dsRNA has not been successful in most cell types because of nonspecific responses elicited by dsRNA molecules longer than about 30 nt (17). However, Tuschl and coworkers (13, 18) recently made the remarkable observation that transfection of synthetic 21-nt siRNA duplexes into mammalian cells effectively inhibits endogenous genes in a sequence-specific manner. These siRNA duplexes are too short to trigger the nonspecific dsRNA responses, but they still cause destruction of complementary RNA sequences (19). It is not known whether siRNAs in mammalian cells also prime synthesis of dsRNA to form additional siRNAs. The recent discovery of large numbers of microRNA genes (reviewed in ref. 20) raises the prospect that the cellular machinery necessary for siRNA inhibition in mammalian cells may be linked to normal processes of gene regulation.In the hope of applying...

Inhibitory efficacy of hypoxia-inducible factor 1α short hairpin RNA plasmid DNA-loaded poly (D, L-lactide-co-glycolide) nanoparticles on choroidal neovascularization in a laser-induced rat model

“…[56][57][58] In addition to siRNA, DNA vectors that transcribe siRNA or shRNA are also available to induce RNAi. [59][60][61] Nonviral vector-based approach using pDNA is expected to be a safer method to induce RNAi compared with any approaches using viral vectors, 62,63 and the vector-based shRNA have evolved as an extremely powerful and the most popular gene-silencing reagent currently. shRNA are more efficient than siRNA on the induction of gene silencing and can be continuously synthesized by the …”

RNA interference: The molecular immune system

“…Reactivation of transposons after the siRNA generating system supports this notion (Ketting et al 1999, Tabara et al 1999 Yu et al 2002). It is not known how the dsRNAs exit the cell in which they are produced, how they are systemically disseminated, or how they are taken up by distant target cells.…”

“…Numerous additional reports indicate that certain individuals or groups are immune to HIV-1 altogether (Baur et al 1989, Bryson et al 1995, Deacon et al 1995, Roques et al 1995, Paxton et al 1996, Kaul et al 2000, Kulkarni et al 2003, while others remain AIDS free for extensive periods of time . In order to develop sufficient human protection against HIV-1 variants, a number of homologous sequences will be required in the endogenous repertoire (Hiroshika et al 2000, Donze & Picard 2002, Hohjoh 2002, Jeong et al 2002, Leirdal & Sioud 2002, Miyagishi & Taira 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002, Yu et al 2002. Through genetic technology, however, a rapid HIV-1 inhibitory siRNAs specific repertoire can perhaps be designed.…”

“…Through genetic technology, however, a rapid HIV-1 inhibitory siRNAs specific repertoire can perhaps be designed. These siRNAs could be encoded and delivered by appropriate vectors and then utilized in preventative or therapeutic vaccines (Hiroshika et al 2000, Donze & Picard 2002, Hohjoh 2002, Jeong et al 2002, Leirdal & Sioud 2002, Miyagishi & Taira 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002, Yu et al 2002.…”

Gene Therapy Progress and Prospects: Recent progress in transgene and RNAi expression cassettes

“…Thus Pol III transcripts normally have 3-4 U residues at the 3 0 end (for a review see Schramm and Hernandez 21 ). A flurry of papers in 2002 from five different groups [22][23][24][25][26] established that short hairpin RNA (shRNA) molecules expressed from U6 or H1 Type 3 Pol III promoters could mediate sequence-specific RNAi-based gene inhibition. All of the groups designed expression units where the RNA sequence expressed from the Pol III promoters contained an inverted repeat of 19-29 nt separated by a short spacer sequence (See Figure 1a).…”