Comparative high-throughput RNAi screening methodologies in C. elegans and mammalian cells

Simpson, Kaylene J.; Davis, Gregory F.; Boag, Peter R.

doi:10.1016/j.nbt.2012.01.003

Cited by 18 publications

(14 citation statements)

References 87 publications

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“…Such screens have proved highly successful in Caenorhabditis elegans, Drosophila melanogaster and mammalian tissue culture models. Mammalian screens have largely focused on the field of cancer biology and have been used to identify genes involved in cell proliferation, survival, invasion and migration, and responses to drugs (reviewed by Simpson et al, 2012). …”

Section: Introductionmentioning

confidence: 99%

Genome-wide shRNA screening to identify factors mediating Gata6 repression in mouse embryonic stem cells

Cooper

Brockdorff

2013

Development

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The use of whole-genome pooled shRNA libraries in loss-of-function screening in tissue culture models provides an effective means to identify novel factors acting in pathways of interest. Embryonic stem cells (ESCs) offer a unique opportunity to study processes involved in stem cell pluripotency and differentiation. Here, we report a genome-wide shRNA screen in ESCs to identify novel components involved in repression of the Gata6 locus, using a cell viability-based screen, which offers the benefits of stable shRNA integration and a robust and simple protocol for hit identification. Candidate factors identified were enriched for transcription factors and included known Polycomb proteins and other chromatin-modifying factors. We identified the protein Bcor, which is known to associate in complexes with the Polycomb protein Ring1B, and verified its importance in Gata6 repression in ESCs. Potential further applications of such a screening strategy could allow the identification of factors important for regulation of gene expression and pluripotency.

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Section: Introductionmentioning

confidence: 99%

Genome-wide shRNA screening to identify factors mediating Gata6 repression in mouse embryonic stem cells

Cooper

Brockdorff

2013

Development

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“…Cette approche est aussi bien utilisée pour des criblages de banques de composés (de 1 000 à plusieurs milliers de composés, petit à haut débit de criblage), que pour les phases de validation et caractérisation des composés bio-actifs sélec-tionnés à l'issue d'un criblage HTS par exemple (étude dose-réponse, études de relation structure-activité, étude de l'effet du composé sur différents phénotypes cellulaires, etc.). Pour aller encore plus loin dans les possibilités offertes par cette technologie, des équipes l'appliquent à des organismes modèles, tels que T. gondii, C. elegans, le zebrafish, ou Drosophila melanogaster [5,[36][37][38][39]. De plus, afin de travailler sur des modèles d'étude in vivo plus proches de la réalité, de nombreux efforts sont réalisés dans la mise en place de modèles en trois dimensions (3D) comme les cultures de sphé-roïdes [40] ou des cocultures en 3D [41].…”

Section: Le Hcs En Recherche Académiqueunclassified

Criblage phénotypique à haut contenu pour la chémobiologie et ses enjeux

2015

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“…1 This has allowed scientists to perform up to genome-scale simultaneous gene knockdowns at an industrial scale against arrayed or pooled RNAi libraries, and with a battery of assay readouts ranging from simple cell metabolic measurements to highly sophisticated deep sequencing methods; as such many mammalian RNAi screens have been conducted to date and reported in various fields with a bias for novel target discovery in cancer biology and host-virus interactions. 2–3 …”

Section: Introductionmentioning

confidence: 99%

Systematic Analysis of RNAi Reports Identifies Dismal Commonality at Gene-Level and Reveals an Unprecedented Enrichment in Pooled shRNA Screens

Bhinder¹,

Djaballah²

2013

CCHTS

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RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes.

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Comparative high-throughput RNAi screening methodologies in C. elegans and mammalian cells

Cited by 18 publications

References 87 publications

Genome-wide shRNA screening to identify factors mediating Gata6 repression in mouse embryonic stem cells

Genome-wide shRNA screening to identify factors mediating Gata6 repression in mouse embryonic stem cells

Criblage phénotypique à haut contenu pour la chémobiologie et ses enjeux

Systematic Analysis of RNAi Reports Identifies Dismal Commonality at Gene-Level and Reveals an Unprecedented Enrichment in Pooled shRNA Screens

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