A resource for large-scale RNA-interference-based screens in mammals

Paddison, Patrick J.; Silva, José M.; Conklin, Douglas S.; Schlabach, Mike; Li, Mamie Z.; Aruleba, Shola; Balija, Vivekanand S.; O’Shaughnessy, Andrew; Gnoj, L.; Scobie, Kim; Chang, Kenneth; Westbrook, Thomas F.; Cleary, Michele A.; Sachidanandam, Ravi; McCombie, W. Richard; Elledge, Stephen J.; Hannon, Gregory J.

doi:10.1038/nature02370

Cited by 643 publications

(444 citation statements)

References 16 publications

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“…However, recently, companies like System Biosciences (Mountain View, CA, USA) and Galapagos Genomics (Mechelen, Belgium) have created selectable libraries consisting of shRNA expression libraries with viral vectors. Hannon and co-workers 97 have developed a large plasmid library coupled to a matrix-assisted genetically integrated cloning technology comprising of atleast 28 000 shRNA cassettes. This technology has been postulated to have the ability to screen at the single cell level.…”

Section: Rnai and Functional Genomicsmentioning

confidence: 99%

siRNA-based approaches in cancer therapy

2006

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The availability of the human genome sequence has revolutionized the strategy of employing nucleic acids with sequences complementary to specific target genes to improve drug discovery and target validation. Development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences offers the possibility of rational design with high specificity, lacking in many current drug treatments for various diseases including cancer, at relatively inexpensive costs. Antisense technology is one such example that has shown promising results and boasts of yielding the only approved drug to date in the genomics field. However, in vivo delivery issues have yet to be completely overcome for widespread clinical applications. In contrast to antisense oligonucleotides, the mechanism of silencing an endogenous gene by the introduction of a homologous double-stranded RNA (dsRNA), transgene or virus is called post-transcriptional gene silencing (PTGS) or RNA interference. PTGS is a natural mechanism whereby metazoan cells suppress expansion of genes when they come across dsRNA molecules with the same sequence. Short interfering RNA is currently the fastest growing sector of this antigene field for target validation and therapeutic applications. Although, in theory, the development of genomics-based agents to inhibit gene expression is simple and straightforward, the fundamental concern relies upon the capacity of the oligonucleotide to gain access to the target RNA. This paper summarizes the advances in the last decade in the field of PTGS using RNA interference approaches and provides relevant comparisons with other oligonucleotide-based approaches with a specific focus on oncology applications. Cancer Gene Therapy (2006) Genomic-based strategiesThe American Cancer Society estimates that, in 2005, a total of 1 372 910 new cancer cases and 570 280 deaths are expected in the United States. 1 These morbid statistics demonstrate the necessity of newer therapeutic modalities for achieving successful cancer treatment and cure. Downregulation of genes that contribute to cancer progression has been the goal of targeted genomics-based strategies, with the expectation that such an approach may lead to selective and specific inhibition of tumor growth with minimal untoward side effects on normal cells. Identification of target genes involved in neoplastic transformation and tumor progression has triggered the idea that nucleotide sequences of cancer-relevant genes could lead to the development of tailored anticancer agents that lack many of the toxic side effects of traditional cytotoxic drugs. This has led to a recent acceleration in the development and optimization of genomic-based strategies for cancer therapy. This idea dates back to the 1960s when RNA sequences were shown to serve as endogenous inhibitors of gene expression in procaryotes. The antigene approaches include the following:(1) Ribozymes: 2,3 These were discovered in the 1970s, and the elucidation of the transactivating hammerh...

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Section: Rnai and Functional Genomicsmentioning

confidence: 99%

siRNA-based approaches in cancer therapy

2006

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“…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 .…”

Section: High-throughput Rnai Screens In Cultured Cellsmentioning

confidence: 99%

Cell microarrays and RNA interference chip away at gene function

2005

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S25 P E R S P E C T I V EThe recent development of cell microarrays offers the potential to accelerate high-throughput functional genetic studies. The widespread use of RNA interference (RNAi) has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technology, RNAi cell microarrays promise to increase the efficiency, economy and ease of genome-wide RNAi screens in metazoan cells.

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“…Others have used an 'external' bar code, in which a separate random 60-mer oligonucleotide is linked in cis to the shRNA vector 17,18 . Note that the oligonucleotides that we spot on the microarray for bar-code hybridizations are not the 19-mer sequences, but the 60-mer oligonucleotides that were used to generate the shRNA library (see Fig.…”

Section: Barcoding Technologymentioning

confidence: 99%