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

Fingerprint

Leishmania
Essential Genes
Gene Deletion
Genes
Enzyme Activation
Sirolimus
Genetic Recombination
Parasites
Cre recombinase

Keywords

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

Cite this

Duncan, S. M., Myburgh, E., Alves-Ferreira, E. V., & Mottram, J. C. (2019). DiCre-Based Inducible Disruption of Leishmania Genes. In J. Clos (Ed.), Leishmania: Methods and Protocols (Vol. 1971, pp. 211-224). (Methods in Molecular Biology; Vol. 1971). New York: Humana Press. https://doi.org/10.1007/978-1-4939-9210-2_10
Duncan, Samuel M. ; Myburgh, Elmarie ; Alves-Ferreira, Eliza V. ; Mottram, Jeremy C. / DiCre-Based Inducible Disruption of Leishmania Genes. Leishmania: Methods and Protocols. editor / Joachim Clos. Vol. 1971 New York : Humana Press, 2019. pp. 211-224 (Methods in Molecular Biology).
@inbook{224f012a9a664a03ba16dea0f586271a,
title = "DiCre-Based Inducible Disruption of Leishmania Genes",
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.",
keywords = "Conditional gene deletion, Cre recombinase, DiCre, Essential genes, Leishmania, LoxP sites, Rapamycin",
author = "Duncan, {Samuel M.} and Elmarie Myburgh and Alves-Ferreira, {Eliza V.} and Mottram, {Jeremy C.}",
note = "This work was supported by the Medical Research Council (MR/K019384) and the Wellcome Trust (104976, 104111).",
year = "2019",
doi = "10.1007/978-1-4939-9210-2_10",
language = "English",
isbn = "9781493992096",
volume = "1971",
series = "Methods in Molecular Biology",
publisher = "Humana Press",
pages = "211--224",
editor = "Joachim Clos",
booktitle = "Leishmania",

}

Duncan, SM, Myburgh, E, Alves-Ferreira, EV & Mottram, JC 2019, DiCre-Based Inducible Disruption of Leishmania Genes. in J Clos (ed.), Leishmania: Methods and Protocols. vol. 1971, Methods in Molecular Biology, vol. 1971, Humana Press, New York, pp. 211-224. https://doi.org/10.1007/978-1-4939-9210-2_10

DiCre-Based Inducible Disruption of Leishmania Genes. / Duncan, Samuel M.; Myburgh, Elmarie; Alves-Ferreira, Eliza V.; Mottram, Jeremy C. (Lead / Corresponding author).

Leishmania: Methods and Protocols. ed. / Joachim Clos. Vol. 1971 New York : Humana Press, 2019. p. 211-224 (Methods in Molecular Biology; Vol. 1971).

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

TY - CHAP

T1 - DiCre-Based Inducible Disruption of Leishmania Genes

AU - Duncan, Samuel M.

AU - Myburgh, Elmarie

AU - Alves-Ferreira, Eliza V.

AU - Mottram, Jeremy C.

N1 - This work was supported by the Medical Research Council (MR/K019384) and the Wellcome Trust (104976, 104111).

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Conditional gene deletion

KW - Cre recombinase

KW - DiCre

KW - Essential genes

KW - Leishmania

KW - LoxP sites

KW - Rapamycin

UR - http://www.scopus.com/inward/record.url?scp=85064769449&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-9210-2_10

DO - 10.1007/978-1-4939-9210-2_10

M3 - Chapter (peer-reviewed)

C2 - 30980305

SN - 9781493992096

VL - 1971

T3 - Methods in Molecular Biology

SP - 211

EP - 224

BT - Leishmania

A2 - Clos, Joachim

PB - Humana Press

CY - New York

ER -

Duncan SM, Myburgh E, Alves-Ferreira EV, Mottram JC. DiCre-Based Inducible Disruption of Leishmania Genes. In Clos J, editor, Leishmania: Methods and Protocols. Vol. 1971. New York: Humana Press. 2019. p. 211-224. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-9210-2_10