High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans

O’Reilly, Linda P.; Knoerdel, Ryan R.; Silverman, Gary A.; Pak, Stephen C.

doi:10.1007/978-1-4939-6337-9_12

Cited by 7 publications

(2 citation statements)

References 21 publications

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“…The integration of automated worm sorting with microscopy platforms that facilitate automated image acquisition, worm segmentation and data analysis has advanced existing genome-wide screening strategies [ 20 – 23 ]. Moreover, the small size and transparency of the worm coupled with systemic RNA interference (RNAi) methodology has facilitated high-throughput and high-content whole animal screenings using fluorescent protein reporters or dyes, as well as drug screening [ 24 – 29 ]. However, sorting of homozygous mutants from a balanced strain has never been used to study genetic interactions of essential genes.…”

Section: Introductionmentioning

confidence: 99%

Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

et al. 2018

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BackgroundAdvances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking.ResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPRmt and growth.ConclusionsThe method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user’s requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0496-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

et al. 2018

View full text Add to dashboard Cite

show abstract

“…The integration of automated worm sorting with microscopy platforms that facilitate automated image acquisition, worm segmentation and data analysis, has advanced existing genome-wide screening strategies [20][21][22][23]. Moreover, the small size and transparency of the worm coupled with the systemic RNAi methodology has facilitated high-throughput and high-content whole animal screenings using fluorescent protein reporters or dyes, as well as drug screening [24][25][26][27][28][29]. However, sorting of homozygous mutants from a balanced strain has never been used to study genetic interactions of essential genes.…”

Section: Introductionmentioning

confidence: 99%