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An arrayed genome-scale lentiviral-enabled short hairpin RNA screen identifies lethal and rescuer gene candidates.

ABSTRACT: RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of candidates toward a better understanding of cellular homeostasis. Using a high-stringency hit nomination method encompassing criteria of at least three active hairpins per gene and filtered for potential off-target effects (OTEs), referred to as the Bhinder-Djaballah analysis method, we identified 1,252 lethal and 6 rescuer gene candidates, knockdown of which resulted in severe cell death or enhanced growth, respectively. Cross referencing individual hairpins with the TRC1 validated clone database, 239 of the 1,252 candidates were deemed independently validated with at least three validated clones. Through our systematic OTE analysis, we have identified 31 microRNAs (miRNAs) in lethal and 2 in rescuer genes; all having a seed heptamer mimic in the corresponding shRNA hairpins and likely cause of the OTE observed in our screen, perhaps unraveling a previously unknown plausible essentiality of these miRNAs in cellular viability. Taken together, we report on a methodology for performing large-scale arrayed shRNA screens, a comprehensive analysis method to nominate high-confidence hits, and a performance assessment of the TRC1 library highlighting the intracellular inefficiencies of shRNA processing in general.

SUBMITTER: Bhinder B

PROVIDER: S-EPMC3619155 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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An arrayed genome-scale lentiviral-enabled short hairpin RNA screen identifies lethal and rescuer gene candidates.

Bhinder Bhavneet B Antczak Christophe C Ramirez Christina N CN Shum David D Liu-Sullivan Nancy N Radu Constantin C Frattini Mark G MG Djaballah Hakim H

Assay and drug development technologies 20121130 3

RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of ...[more]

PMID: 23198867

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Project description:Reactive oxygen species are important elements in ototoxic damage to hair cells (HCs), appearing early in the damage process. Higher levels of natural antioxidants are positively correlated with resistance to ototoxins and many studies have shown that exogenous antioxidants can protect HCs from damage. While a very wide variety of antioxidants with different characteristics and intracellular targets exist, most ototoxicity studies have focused upon one or a few well-characterized compounds. Relatively little research has attempted to determine the comparative efficacy of large variety of different antioxidants. This has been in part due to the lack of translation between cell culture and in vivo measures of efficacy. To circumvent this limitation, we used an in vitro assay based on micro-explants from the basal and middle turns of the neonatal mouse organ of Corti to screen a commercial redox library of diverse antioxidant compounds for their ability to protect mammalian HCs from a high dose of the ototoxic antibiotic gentamicin. The library included several antioxidants that have previously been studied as potential treatments for HC damage, as well as many antioxidants that have never been applied to ototoxicity. The micro-explants were treated with 200 ?M gentamicin alone, gentamicin plus one of three dosages of a redox compound, the highest dosage of compound alone, or were untreated. HC counts were determined before the gentamicin insult and at 1, 2, and 3 days afterward to evaluate the HC survival. From a total of 81 antioxidant compounds, 13 exhibited significant protection of HCs. These included members of a variety of antioxidant classes with several novel antioxidants, not previously tested on HCs, appearing to alleviate the damaging gentamicin effect. Some compounds previously shown to be protective of HCs were correspondingly protective in this in vitro screen, while others were not. Finally, one of the three pro-oxidant compounds included in the library as well as six antioxidants exhibited evidence of toxicity in the absence of gentamicin. The results demonstrate the wide variability in the ability of antioxidants to protect HCs from high-dose gentamicin damage, and identify promising candidate leads for further study as potential drug targets.Highlights• A medium-throughput assay based on micro-explants of the organ of Corti was developed to screen mammalian cochlear hair cells for protection from damage by ototoxins.• Eighty one antioxidants and 3 pro-oxidants were evaluated for hair cell protection from high-dose gentamicin.• Thirteen antioxidants were significantly protective, while 6 proved to be damaging.• The use of a common assay permitted an evaluation of the relative capacity of different antioxidants for the protection of hair cells.

Dataset Information

An arrayed genome-scale lentiviral-enabled short hairpin RNA screen identifies lethal and rescuer gene candidates.

Publications

An arrayed genome-scale lentiviral-enabled short hairpin RNA screen identifies lethal and rescuer gene candidates.

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