A DNA vector-based RNAi technology to suppress gene expression in mammalian cells

Abstract

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic doublestranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells. D ouble-stranded RNA (dsRNA) can trigger silencing of homologous gene expression by a mechanism termed RNAi (for RNA-mediated interference) (1). RNAi is an evolutionarily conserved phenomenon and a multistep process that involves generation of active small interfering RNA (siRNA) in vivo through the action of an RNase III endonuclease, Dicer. The resulting 21-to 23-nt siRNA mediates degradation of the complementary homologous RNA (reviewed in refs. 2-4). RNAi has been used as a reverse genetic tool to study gene function in multiple model organisms, including plants, Caenorhabditis elegans, and Drosophila where large dsRNAs efficiently induce gene-specific silencing (1, 5-7).One obstacle to achieving RNAi in mammals is that dsRNAs longer than 30 nt will activate an antiviral response, leading to the nonspecific degradation of RNA transcripts and a general shutdown of host cell protein translation (8, 9). As a result, the long dsRNA, with a few exceptions (10, 11), does not produce RNAi activity, and RNAi therefore is not a general method for silencing specific genes in mammalian cells. This obstacle has been recently overcome by Tuschl and colleagues (12) who found that gene-specific suppression in mammalian cells can be achieved by vitro-synthesized siRNA that are 21 nt in length, long enough to induce gene-specific suppression, but short enough to evade the host interferon response.In this article, we describe a DNA vector-based approach to achieve RNAi in mammalian cells. With this approach, small RNAs are predicted to be synthesized from a DNA template under the control of an RNA polymerase III (Pol III) promoter in transfected cells. Pol III has the advantage of directing the synthesis of small, noncoding transcripts whose 3Ј ends are defined by termination within a stretch of 4-5 thymidines (Ts) (13). These properties make it possible to use DNA templates to synthesize, in vivo, small RNAs with structural features close to what has been found to be required for active siRNAs synthesized in vitro (14).Using this DNA vector-based RNAi approach, we show that transfected as well as endogenous genes can be efficiently inhibited. We have examined the effects of the in vivosynthe...

Oncolytic adenovirus-mediated shRNA against Apollon inhibits tumor cell growth and enhances antitumor effect of 5-fluorouracil

“…35 Three siRNA sequences from various coding regions of human Apollon gene were selected (Figure 1b). To generate an intermediate plasmid for cloning shRNAs targeted to Apollon, the oligonucleotide 5 0 -GGCCAGTATGGCTTGCTAGTA-3 0 annealed with the oligonucleotide 5 0 -AGCTTACTAGCAAGCCA TACTGGCC-3 0 was inserted into the ApaI (blunted) and HindIII sites of pBS/U6.…”

RNA interference: The molecular immune system

“…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.…”

“…One way to answer this question is to evaluate what happens to an organism when certain genes, presumed to be involved in RBGS, are altered. Mutations in several genes render an organism incapable of generating siRNAs; in some cases, these mutations reactivate transposons and retroelements (Ketting et al 1999, Tabara et al 1999, Hirochika et al 2000, Nakayashiki et al 2001, Jeong et al 2002, Paddison et al 2002, Paul et al 2002, Sui et al 2002. Proteins essential for the homologous RNA degradation pathway are highly conserved in fungi, plants, vertebrates, and mammals (reviewed in Grishok & Mello 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).…”