Gene Replacement by Homologous Recombination
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 wordsLeishmania Homologous recombination Gene replacement Reverse genetics
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.
- 5.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.13375CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Ubeda JM, Raymond F, Mukherjee A, Plourde M, Gingras H, Roy G, Lapointe A, Leprohon P, Papadopoulou B, Corbeil J, Ouellette M (2014) Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol 12(5):e1001868. https://doi.org/10.1371/journal.pbio.1001868CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Sambrook J, Russell DW (2001) Molecular Cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar