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Leishmania pp 211-224 | Cite as

DiCre-Based Inducible Disruption of Leishmania Genes

  • Samuel M. Duncan
  • Elmarie Myburgh
  • Eliza V. Alves-Ferreira
  • Jeremy C. MottramEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1971)

Abstract

Conditional gene deletion using dimerizable Cre recombinase (DiCre) is so far the best developed system for the phenotypic analysis of essential genes in Leishmania species. Here, we describe a protocol for the generation of a conditional gene deletion mutant and the subsequent inducible deletion of a target gene. Leishmania parasites are genetically modified to express two inactive Cre subunits (DiCre) and a single LoxP-flanked version of a target gene in a context where both endogenous copies of the gene have been deleted. Treatment with rapamycin dimerizes the DiCre subunits, resulting in activation of the enzyme, recombination between the LoxP sites, and excision of the LoxP-flanked target gene. Subsequent phenotyping allows for the analysis of essential gene function.

Key words

DiCre Cre recombinase Conditional gene deletion Essential genes LoxP sites Rapamycin Leishmania 

Notes

Acknowledgments

This work was supported by the Medical Research Council (MR/K019384) and the Wellcome Trust (104976, 104111).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Samuel M. Duncan
    • 1
  • Elmarie Myburgh
    • 2
  • Eliza V. Alves-Ferreira
    • 2
  • Jeremy C. Mottram
    • 2
    Email author
  1. 1.Division of Biological Chemistry and Drug Discovery, Wellcome Trust Centre for Anti-infectives ResearchUniversity of DundeeDundeeUK
  2. 2.Department of Biology, York Biomedical Research InstituteUniversity of YorkYorkUK

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