Functional genomics with the use of RNA interference libraries

RNA interference (RNAi) has revolutionised functional genomics allowing selective silencing of individual genes and rapid assignment of gene function through administration or expression of short interfering RNA (siRNA). To make use of the power of this technology and to enable high throughput target identification and validation in mammalian cells, we have designed an RNA interference library that targets approximately 8000 human genes. This large set of genes has been selected on the association with cancer and other human diseases. This collection of genes represents components of major cellular pathways including cell cycle, transcription regulation, stress signalling, signal transduction and important biological processes such as biosynthesis, proteolysis and metabolism. In addition, target genes in the library have been selected on the basis of druggability and include gene families such as kinases and GPCRs.

The human SUPER RNAi™ library is based on the pSUPER RNAi system developed by Brummelkamp et al. This system uses a mammalian expression vector that directs the synthesis of short interfering RNA (siRNA)-like transcripts. The vector contains the polymerase III Histone H1-RNA promoter that produces a small RNA transcript with a defined start of transcription and a termination signal of five thymidines in a row (T5). The transcript is terminated after the second uridine resulting in a transcript that forms a hairpin structure with two T or U nucleotides at the 3' end. The hairpin transcript is processed in the cell into a 21 nucleotide siRNA. These siRNAs can induce a strong and specific suppression of gene expression. The stable expression of siRNAs using the pSUPER vector can mediate suppression of gene expression over prolonged periods of time. This allows analysis of loss-of-function phenotypes in long term assays.

The human SUPER RNAi™ library has many applications including powerful loss of function bar-code screens. The bar-code technique, pioneered in yeast, allows for the quantitative analyses of large numbers of shRNA vectors in one population. In each shRNA vector in the human SUPER RNAi™ library is contained a unique identifier (barcode), the gene specific 19-mer siRNA sequence. This sequence can be used to asses the relative abundance of each shRNA vector by hybridization to a DNA micro-array, compromising all 24.000 shRNA sequences present in the human SUPER RNAi™ library. The bar-code application will facilitate rapid screening of large pools of shRNA vectors and will allow for the identification of shRNA vectors that are selected against in the population either presence or absence of a specific treatment. The latter will allow us to perform synthetic lethality screens in human cells.

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References

• Berns, K., Hijmans, E. M., Mullenders, J., Brummelkamp, T. R., Velds, A., Heimerikx, M., Kerkhoven, R. M., Madiredjo, M., Nijkamp, W., Weigelt, B., et al. (2004). A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature in press.
• Brummelkamp, T. R., Bernards, R., and Agami, R. (2002a). Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243-247.
• Brummelkamp, T. R., Bernards, R., and Agami, R. (2002b). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.
• Brummelkamp TR, Bernards R. (2003) New tools for functional mammalian cancer genetics. Nat Rev Cancer 3 (10), 781-789.
• Brummelkamp TR, Nijman SM, Dirac AM, Bernards R. (2003) Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature 424, 797-801.