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Malaria pp 127-138 | Cite as

Recombination-Mediated Genetic Engineering of Plasmodium berghei DNA

  • Claudia Pfander
  • Burcu Anar
  • Mathieu Brochet
  • Julian C. Rayner
  • Oliver BillkerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 923)

Abstract

DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.

Key words

Gene targeting Recombineering Genomic DNA library Gateway cloning 

References

  1. 1.
    Pfander C et al (2011) A scalable pipeline for highly effective genetic modification of a malaria parasite. Nat Methods 8:1078–1082Google Scholar
  2. 2.
    Ravin NV, Ravin VK (1999) 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:e13PubMedCrossRefGoogle Scholar
  3. 3.
    Godiska R et al (2010) Linear plasmid ­vector for cloning of repetitive or unstable sequences in Escherichia coli. Nucleic Acids Res 38:e88PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang Y et al (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128PubMedCrossRefGoogle Scholar
  5. 5.
    Poser I et al (2008) BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 5:409–415PubMedCrossRefGoogle Scholar
  6. 6.
    Sarov M et al (2006) A recombineering pipeline for functional genomics applied to Caenorha­bditis elegans. Nat Methods 3:839–844PubMedCrossRefGoogle Scholar
  7. 7.
    Skarnes WC et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342PubMedCrossRefGoogle Scholar
  8. 8.
    Braks JA et al (2006) Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium. Nucleic Acids Res 34:e39PubMedCrossRefGoogle Scholar
  9. 9.
    Wang J, Sarov M, Rientjes J, Fu J, Hollak H, Kranz H, Xie W, Stewart AF, Zhang Y (2006) An improved recombineering approach by adding RecA to lambda Red recombination. Mol Biotechnol 32:43–53PubMedCrossRefGoogle Scholar
  10. 10.
    Janse CJ et al (2006) High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc 1:346–356PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Claudia Pfander
    • 1
  • Burcu Anar
    • 1
  • Mathieu Brochet
    • 1
  • Julian C. Rayner
    • 1
  • Oliver Billker
    • 1
    Email author
  1. 1.Wellcome Trust Sanger InstituteCambridgeUK

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