An approach to genomewide screens of expressed small interfering RNAs in mammalian cells

Abstract

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a genespecific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3 terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8,000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-B signaling pathway. RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is suppressed by the introduction of homologous double-stranded RNAs (dsRNAs). After dsRNA molecules are delivered to the cytoplasm of a cell, they are cleaved by the RNase III-like enzyme, Dicer, to 21-to 23-nt small interfering RNAs (siRNAs) (1). These siRNAs are then incorporated into a protein complex, the RNA-induced silence complex (RISC). The antisense strand of the duplex siRNA guides the RISC to the homologous mRNA, where the RISC-associated endoribonuclease cleaves the target mRNA, resulting in silencing of the target gene (2). RNAi has been successfully used to suppress gene expression in a variety of organisms including zebrafish, Caenorhabditis elegans, Drosophila, planaria, mice, and mammalian cells (3,4). In C. elegans and Drosophila, RNAi is typically induced by the introduction of a long dsRNA (up to 1-2 kb) produced by in vitro transcription. This simple approach cannot be used in mammalian cells, where introduction of long dsRNA elicits a strong antiviral response that obscures any gene-specific silencing effect. Much of this response is caused by activation of the dsRNA-dependent protein kinase PKR, which phosphorylates and inactivates the translation initiation factor eIF2a (5, 6). However, introduction of 21-nt siRNAs into mammalian cells does not stimulate the antiviral response in mammalian cells and can effectively target specific mRNAs, resulting in gene silencing (7).Short siRNA molecules can be prepared by chemical synthesis or in vitro transcription. Alternatively, they can be transcribed in vivo by using siRNA expression vectors with a RNA polymerase (pol) III promoter (including U6, human H1, and tRNA promoters) (8, 9), or a pol II promoter with a minimal poly(A) signal sequence (10). Tissue-specific pol II promot...

Cell microarrays and RNA interference chip away at gene function

“…Instead of generating large libraries of chemically synthesized siRNAs, several investigators have created their own large-scale shRNA or siRNA libraries encoded by plasmid vectors and used these libraries to identify new genes involved in the p53 pathway 33 , human proteasome function 34 and the NF-κB signaling pathway 35 . These screens demonstrate the utility of large RNAi screens in mammalian cells but also underscore the need for better siRNA and shRNA selection methods because many RNAi constructs that should have elicited effects did not 35 . A development that may help with this issue is the use of endonuclease-prepared siRNAs, siRNAs that are generated by the RNase III-mediated degradation of long dsRNAs 36 .…”

RNA interference and potential therapeutic applications of short interfering RNAs

“…59,60 In those studies, sets of arrayed expression libraries from chemically synthesized oligonucleotides which target 7914 and 9610 different human genes, respectively, were produced. 59,60 Other approaches for large-scale genome-wide screening using RNAi, which involve generating a library of polymerase chain reaction products that encode shRNAs 61 and constructing random shRNA libraries based on manipulation of complementary or genomic DNA, [62][63][64] have also been reported recently. RNAi using genome-wide libraries is expected to complement current experiments that aim to functionally map the mouse genome by chemical or insertional mutagenesis.…”

RNAi and HTS: exploring cancer by systematic loss-of-function

“…Initial proof of concept for RNAi screening using both synthetic siRNAs and plasmid-encoded shRNAs were reported last year using gene-family focused libraries Brummelkamp et al, 2003). In rapid succession, Zheng et al (2004) reported a PCR-based approach to generate a DNA-mediated RNAi library representing >8000 human genes and Berns et al (2004) described the construction and application of a retroviral library encoding shRNAs covering 7194 targets. Finally, Paddison et al (2004) reported the creation of a retroviral library encoding shRNAs targeted against 10 000 human and 5000 mouse genes.…”

“…In addition, no selection is required and both activators and repressors can be detected in the same experiment. This format has been used successfully in both RNA-and DNA-based screens Zheng et al, 2004). However, this format requires considerable up-front effort, including the design, synthesis and arraying of the individual reagents.…”