Abstract
BackgroundGenomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology.ResultsWe describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified.ConclusionsWe present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.
Highlights
Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge
We describe an adaptation of Tn-seq [3], restriction enzyme-mediated integration (REMI)-seq, that allows high throughput parallel identification of REMI insertion points and its use to generate near genome-wide resources which have been made freely available for the D. discoideum research community
REMI can be used to generate mutations in most D. discoideum genes Genome-wide collections of mutants are useful for researchers seeking either to ascribe gene function by reverse genetics experiments on individual clones or unbiased forward genetic screens on pools of mutants
Summary
Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Our ability to comprehensively ascribe function to gene sequences is still limited. Efficient tools for random or systematic genome-wide mutagenesis are crucial and major efforts have been made to develop these tools in model organisms. Genome-wide functional analyses are only possible in a handful of higher eukaryotes. This is because large diploid genomes provide an obstacle to generating extensive mutant collections. Phenotypic analyses are experimentally challenging as individual strains often must be grown up and examined. Whilst libraries of cell culture mutants provide a potential solution, this in turn limits the spectrum of phenotypes that can be studied
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
