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Innovations in Interference

By Robert M. Frederickson

Nov 15, 2005 | RNA interference (RNAi) has been rapidly transformed from newly discovered phenomenon to become one of the most widely used tools in biomedical research. Results of a small clinical trial recently presented by Sirna Therapeutics have demonstrated safety and efficacy of the first small interfering RNA (siRNA)-based drug, Sirna-027. Thus, RNAi has moved from phenomenon to promising drug in less than five years.

However, a perhaps more important role for RNAi is not as a drug itself, but rather as a tool in drug discovery, including target identification and validation. The ability of siRNAs to inhibit gene expression in a specific fashion has led to the creation of an entirely new industry of synthetic RNA manufacturing. The most popular way to trigger RNAi in mammalian cells involves the use of synthetic siRNAs delivered by cationic lipids. It has been shown more recently that siRNAs expressed from eukaryotic promoters and short hairpin RNAs (shRNAs, subsequently processed into siRNAs) could also activate RNAi. A host of companies now offer siRNA and shRNA libraries, custom services, and various delivery formats.

Late last year, a public-private consortium was established to develop and validate tools and methods to facilitate the use of RNAi by the scientific community. Dubbed the RNAi Consortium (TRC), the TRC is creating genome-scale shRNA libraries and developing the methodology to effectively apply these libraries in cell-based screens. Consortium member Sigma-Aldrich has an exclusive agreement with the TRC to distribute the libraries and reagents.

This October, Sigma expanded its MISSION TRC shRNA library product lines to include lentiviral transduction particles in addition to the plasmid DNA and frozen bacterial glycerol stock format. Lentivirus-mediated gene transduction results in higher expression efficiency and greater success with cell types that are refractory to transfection and permits integration of the construct into the genome for long-term shRNA expression.

Going Global
The Genome-Wide RNAi Global Initiative is a separate effort of leading international biomedical research centers that includes a commercial partner and sponsor, Dharmacon, a business unit within Fisher Biosciences (see Dharmacon, Institutes Form siRNA Global Initiative, page 10). Their stated goal is to accelerate the scientific and medical discoveries made possible by the recent availability of the first complete siRNA library to target genes across the entire human genome. Similarly to the TCR, the RNAi Global Initiative will provide a forum for member institutions to share research protocols, establish experimental standards, and develop mechanisms for exchanging and comparing screening data.

A number have announced their intention to house the genomewide siRNA library in central high-throughput screening facilities to increase its availability throughout their organizations.

Dharmacon’s siARRAY Human Genome siRNA Library consists of preselected groups of SMARTselection designed siRNA reagents, individual duplexes, or SMARTpool reagents targeting all unique human genes in the NCBI RefSeq database. The reagents are arranged in 96-well plates for easy storage and rapid preparation and for compatibility with high-throughput liquid handling systems. Dharmacon’s siGENOME claims to offer guaranteed silencing reagents for all unique human, mouse, and rat genes.

Last July, Ambion announced that MitoCheck, an E.U.-funded research consortium, will use the genomewide human siRNA library produced by Ambion in experiments designed to answer fundamental questions of how cell division is regulated. Also in July, Ambion released Silencer CellReady siRNA Libraries, pre-plated siRNAs in the appropriate quantity for use in a single experiment and in a ready-to-transfect format. Current sets target human kinases, the “druggable” genome, and two groups of “popular genes.”

Earlier this year, Ambion introduced the pSilencer 5.1 Retro System, a retrovirus-based expression system that enables researchers to study the long-term effects of gene knock down in cell culture models, including difficult-to-transfect primary cells. Gene expression is reduced through the stable expression of an shRNA using an H1 or U6 promoter.

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