Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A scalable pipeline for highly effective genetic modification of a malaria parasite

Nature Methods volume 8, pages 1078–1082 (2011)Cite this article

Subjects

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.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Characterization of the P. berghei large insert genomic DNA library PbG01.
Figure 2: Modification of PbG01 inserts in E. coli by lambda Red recombineering and site-specific recombinase.
Figure 3: Knock-out vector production in 96 parallel liquid cultures.
Figure 4: Validation of recombineered vectors in P. berghei ANKA.
Figure 5: Effect of homology arm length on targeting frequency.

Similar content being viewed by others

References

  1. Crabb, B.S. & Cowman, A.F. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 93, 7289–7294 (1996).

    Article  CAS  Google Scholar 

  2. van Dijk, M.R., Janse, C.J. & Waters, A.P. Expression of a Plasmodium gene introduced into subtelomeric regions of Plasmodium berghei chromosomes. Science 271, 662–665 (1996).

    Article  CAS  Google Scholar 

  3. Wu, Y., Kirkman, L.A. & Wellems, T.E. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc. Natl. Acad. Sci. USA 93, 1130–1134 (1996).

    Article  CAS  Google Scholar 

  4. Tewari, R. et al. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 8, 377–387 (2010).

    Article  CAS  Google Scholar 

  5. Maier, A.G. et al. Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48–61 (2008).

    Article  CAS  Google Scholar 

  6. Janse, C.J., Ramesar, J. & Waters, A.P. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat. Protoc. 1, 346–356 (2006).

    Article  CAS  Google Scholar 

  7. Hasty, P., Rivera-Perez, J. & Bradley, A. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell. Biol. 11, 5586–5591 (1991).

    Article  CAS  Google Scholar 

  8. Shulman, M.J., Nissen, L. & Collins, C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol. Cell. Biol. 10, 4466–4472 (1990).

    Article  CAS  Google Scholar 

  9. Gardner, M.J. et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511 (2002).

    Article  CAS  Google Scholar 

  10. Hall, N. et al. A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science 307, 82–86 (2005).

    Article  CAS  Google Scholar 

  11. Ejsmont, R.K., Sarov, M., Winkler, S., Lipinski, K.A. & Tomancak, P. A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nat. Methods 6, 435–437 (2009).

    Article  CAS  Google Scholar 

  12. Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).

    Article  CAS  Google Scholar 

  13. Sarov, M. et al. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat. Methods 3, 839–844 (2006).

    Article  CAS  Google Scholar 

  14. Ravin, N.V. & Ravin, V.K. Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures. Nucleic Acids Res. 27, e13 (1999).

    Article  CAS  Google Scholar 

  15. Ravin, N.V., Kuprianov, V.V., Gilcrease, E.B. & Casjens, S.R. Bidirectional replication from an internal ori site of the linear N15 plasmid prophage. Nucleic Acids Res. 31, 6552–6560 (2003).

    Article  CAS  Google Scholar 

  16. Godiska, R. et al. Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli. Nucleic Acids Res. 38, e88 (2010).

    Article  Google Scholar 

  17. Zhang, Y., Buchholz, F., Muyrers, J.P. & Stewart, A.F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    Article  CAS  Google Scholar 

  18. Wang, J. et al. An improved recombineering approach by adding RecA to lambda Red recombination. Mol. Biotechnol. 32, 43–53 (2006).

    Article  Google Scholar 

  19. Gubbels, M.J. et al. Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii. PLoS Pathog. 4, e36 (2008).

    Article  Google Scholar 

  20. Brooks, C.F. et al. The Toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival. Cell Host Microbe 7, 62–73 (2010).

    Article  CAS  Google Scholar 

  21. Skarnes, W.C. et al. A conditional knockout resource for genome-wide analysis of mouse gene function. Nature 474, 337–342 (2011).

    Article  CAS  Google Scholar 

  22. Aurrecoechea, C. et al. PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res. 37, D539–D543 (2009).

    Article  CAS  Google Scholar 

  23. Braks, J.A., Franke-Fayard, B., Kroeze, H., Janse, C.J. & Waters, A.P. Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium. Nucleic Acids Res. 34, e39 (2006).

    Article  Google Scholar 

  24. Menard, R. & Janse, C.J. Gene targeting in malaria parasites. Methods 13, 148–157 (1997).

    Article  CAS  Google Scholar 

  25. Le Roch, K.G. et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503–1508 (2003).

    Article  CAS  Google Scholar 

  26. Tremp, A.Z. & Dessens, J.T. Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape. J. Biol. Chem. 286, 5383–5391 (2011).

    Article  CAS  Google Scholar 

  27. Moon, R.W. et al. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite. PLoS Pathog. 5, e1000599 (2009).

    Article  Google Scholar 

  28. Janse, C.J., Franke-Fayard, B. & Waters, A.P. Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat. Protoc. 1, 614–623 (2006).

    Article  CAS  Google Scholar 

  29. Rutherford, K. et al. Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945 (2000).

    Article  CAS  Google Scholar 

  30. Zerbino, D.R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).

    Article  CAS  Google Scholar 

  31. Assefa, S., Keane, T.M., Otto, T.D., Newbold, C. & Berriman, M. ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics 25, 1968–1969 (2009).

    Article  CAS  Google Scholar 

  32. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  33. Janse, C.J. et al. High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol. Biochem. Parasitol. 145, 60–70 (2006).

    Article  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK

    Claudia Pfander, Burcu Anar, Frank Schwach, Thomas D Otto, Mathieu Brochet, Katrin Volkmann, Michael A Quail, Arnab Pain, Barry Rosen, William Skarnes, Julian C Rayner & Oliver Billker

  2. Chemical Life Sciences and Engineering Division, Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia

    Arnab Pain

Authors
  1. Claudia Pfander
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Burcu Anar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frank Schwach
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas D Otto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mathieu Brochet
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katrin Volkmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael A Quail
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Arnab Pain
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Barry Rosen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. William Skarnes
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Julian C Rayner
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Oliver Billker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Julian C Rayner or Oliver Billker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1–2, Supplementary Protocols 1–3 (PDF 554 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1742

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing