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Leishmania pp 169-188 | Cite as

Gene Replacement by Homologous Recombination

  • Henner Zirpel
  • Joachim ClosEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1971)

Abstract

While homologous recombination-based gene replacement is about to be supplanted by more modern approaches, it is still retaining usefulness for genes that prove to be poor targets for CRISPR/cas-based approaches. Homologous recombination has proven to be relatively robust to minor sequence mismatches between GOI-flanking sequences and the gene replacement constructs, and the faithfulness of recombination events is easily verified by whole-genome sequencing. Moreover, the availability of custom synthetic gene production by numerous service providers should allow for a relatively quick generation of null mutants without the need to introduce additional protein-coding genes beyond the selection markers.

Key words

Leishmania Homologous recombination Gene replacement Reverse genetics 

Notes

Acknowledgments

We are grateful to laboratory alumni Andreas Hübel, Sylvia Krobitsch, Gabi Ommen, Katharina Bartsch, Eugenia Bifeld, and Antje Hombach for their contributions to the refinement of the homologous gene recombination strategy in the laboratory.

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Copyright information

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

Authors and Affiliations

  1. 1.Bernhard Nocht Institute for Tropical MedicineHamburgGermany

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