Abstract
Insertional mutagenesis and depletion (iMAD) is a genetic screening strategy for dissecting complex interactions between two organisms. The simultaneous genetic manipulation of both organisms allows the identification of aggravating and alleviating genetic interactions between pairs of gene disruptions, one from each organism. Hierarchial clustering and genetic interaction networks are then used to identify common behavioral patterns among subsets of genes, which allow functional relationships between proteins and their component pathways to be elucidated. Here we present a protocol for dissecting the interaction between a pathogen (Legionella pneumophila) and its host (cultured Drosophila melanogaster cells) using bacterial mutagenesis and host RNAi. The key stages covered in the PROCEDURE include the design, execution and data analysis of an iMAD screen; details for determining the abundance of individual mutants by microarray analysis and next-generation sequencing are not included because detailed protocols have been published elsewhere. Adapting and optimizing iMAD to a specific experimental system can require 6–18 months. Once a bacterial mutant library, host cell factor depletion strategies and conditions to monitor the interaction are established, an iMAD screen can be completed in 4–8 weeks, depending on the organisms' growth rates, the duration of the interaction and the types of data analysis performed.
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Acknowledgements
This work was supported by the Howard Hughes Medical Institute and a Natalie Zucker Fellowship to T.J.O. R.R.I. is a Howard Hughes Investigator.
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Both authors wrote the paper and designed the experiments. T.J.O. conducted the experiments.
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O'Connor, T., Isberg, R. iMAD, a genetic screening strategy for dissecting complex interactions between a pathogen and its host. Nat Protoc 9, 1916–1930 (2014). https://doi.org/10.1038/nprot.2014.133
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DOI: https://doi.org/10.1038/nprot.2014.133
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