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

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

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

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. © 2011 Nature America, Inc. All rights reserved.
Original languageEnglish (US)
Pages (from-to)1078-1082
Number of pages5
JournalNature Methods
Volume8
Issue number12
DOIs
StatePublished - Oct 23 2011

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Biotechnology

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