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Nature Research, Nature Methods, 12(8), p. 1078-1082, 2011

DOI: 10.1038/nmeth.1742

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

Journal article published in 2011 by Claudia Pfander, Burcu Anar, Frank Schwach, Thomas D. Otto, Mathieu Brochet ORCID, Katrin Volkmann, Michael A. Quail, Arnab Pain, Barry Rosen, William Skarnes, Julian C. Rayner, Oliver Billker ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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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.