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
Part of the Methods in Molecular Biology book series (MIMB, volume 923)


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 


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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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