DiCre-Based Inducible Disruption of Leishmania Genes

Samuel M. Duncan, Elmarie Myburgh, Eliza V. Alves-Ferreira, Jeremy C. Mottram (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

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.

Original languageEnglish
Title of host publicationLeishmania
Subtitle of host publicationMethods and Protocols
EditorsJoachim Clos
Place of PublicationNew York
PublisherHumana Press
Pages211-224
Number of pages14
Volume1971
ISBN (Electronic)9781493992102
ISBN (Print)9781493992096
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
Volume1971
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

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

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