Abstract
RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-α)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators.
Highlights
The introduction of double-stranded RNA into a range of organisms induces both a potent and specific gene silencing effect termed RNA interference (RNAi) [1]. These dsRNAs are processed by Dicer to produce 21–23 nucleotide duplex small interfering RNAs with 2 nucleotide 39 OH overhangs that act as the effectors of gene silencing [2]
In a previous study we reported on the use of convergent transcription from two U6 RNA polymerase III promoters in a plasmid-based system to induce RNAi-mediated gene silencing in mammalian cells [10]
In this paper we describe the construction and characterisation of a random small RNA expression library and demonstrate the utility of this library for identifying small RNA effectors that alter complex cellular phenotypes
Summary
The introduction of double-stranded RNA (dsRNA) into a range of organisms induces both a potent and specific gene silencing effect termed RNA interference (RNAi) [1]. These dsRNAs are processed by Dicer to produce 21–23 nucleotide duplex small interfering RNAs (siRNA) with 2 nucleotide 39 OH overhangs that act as the effectors of gene silencing [2]. It has been demonstrated that chemically synthesised 21 bp siRNAs can be used to induce gene silencing in mammalian cells [3,4]. Convergent transcription-induced RNAi has been demonstrated to be an effective way of controlling specific gene expression in mammalian cells [10,11]
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