Skip to main content
Book cover

Leishmania pp 211–224Cite as

DiCre-Based Inducible Disruption of Leishmania Genes

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

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-9210-2_10
  • Chapter length: 14 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   169.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-9210-2
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Cruz AK, Titus R, Beverley SM (1993) Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc Natl Acad Sci U S A 90(4):1599–1603

    CAS  CrossRef  Google Scholar 

  2. Mottram JC, McCready BP, Brown KG, Grant KM (1996) Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana. Mol Microbiol 22(3):573–583. https://doi.org/10.1046/j.1365-2958.1996.00136.x

    CAS  CrossRef  PubMed  Google Scholar 

  3. Hassan P, Fergusson D, Grant KM, Mottram JC (2001) The CRK3 protein kinase is essential for cell cycle progression of Leishmania mexicana. Mol Biochem Parasitol 113(2):189–198

    CAS  CrossRef  Google Scholar 

  4. Agron PG, Reed SL, Engel JN (2005) An essential, putative MEK kinase of Leishmania major. Mol Biochem Parasitol 142(1):121–125. https://doi.org/10.1016/j.molbiopara.2005.03.007

    CAS  CrossRef  PubMed  Google Scholar 

  5. Duncan SM, Jones NG, Mottram JC (2017) Recent advances in Leishmania reverse genetics: manipulating a manipulative parasite. Mol Biochem Parasitol 216:30–38. https://doi.org/10.1016/j.molbiopara.2017.06.005

    CAS  CrossRef  PubMed  Google Scholar 

  6. Jones NG, Catta-Preta CMC, Lima A, Mottram JC (2018) Genetically validated drug targets in Leishmania: current knowledge and future prospects. ACS Infect Dis 4(4):467–477. https://doi.org/10.1021/acsinfecdis.7b00244

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Jullien N, Sampieri F, Enjalbert A, Herman JP (2003) Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res 31(21):e131

    CrossRef  Google Scholar 

  8. Duncan SM, Myburgh E, Philipon C, Brown E, Meissner M, Brewer J, Mottram JC (2016) Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation. Mol Microbiol 100(6):931–944. https://doi.org/10.1111/mmi.13375

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

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

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jeremy C. Mottram .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

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

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Duncan, S.M., Myburgh, E., Alves-Ferreira, E.V., Mottram, J.C. (2019). DiCre-Based Inducible Disruption of Leishmania Genes. In: Clos, J. (eds) Leishmania. Methods in Molecular Biology, vol 1971. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9210-2_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9210-2_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9209-6

  • Online ISBN: 978-1-4939-9210-2

  • eBook Packages: Springer Protocols