Abstract
In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15–based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
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Acknowledgements
We thank F. Stewart (Technische Universität Dresden) for sharing plasmids pSC101gbdA and pR6K attR1-zeo-PheS-attR2, C. Janse (Leiden University Medical Center) and A. Waters (University of Glasgow) for sharing P. berghei clones RmGM-7 and RmGM-29 and plasmid pL0035, and J. Warren and H. Wang for helping to create a Distributed Annotation System source of clone data for PlasmoDB. This work was supported by the Wellcome Trust (WT089085/Z/09/Z), the Medical Research Council (G0501670) and the European Virtual Institute for Malaria Research Network of Excellence (242095).
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J.C.R and O.B. initiated and directed the research. C.P., A.P., B.R., W.S., J.C.R. and O.B. designed experiments. F.S., T.D.O., M.A.Q. and A.P. generated, sequenced, mapped and quality controlled the PbG01 library. C.P. and B.A. carried out experiments to develop the recombineering pipeline. C.P., M.B. and K.V. carried out experiments to validate the vectors. C.P. and O.B. wrote the manuscript. All authors analyzed data and edited the manuscript.
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Pfander, C., Anar, B., Schwach, F. et al. A scalable pipeline for highly effective genetic modification of a malaria parasite. Nat Methods 8, 1078–1082 (2011). https://doi.org/10.1038/nmeth.1742
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DOI: https://doi.org/10.1038/nmeth.1742
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