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

Handle URI:
http://hdl.handle.net/10754/325358
Title:
A scalable pipeline for highly effective genetic modification of a malaria parasite
Authors:
Pfander, Claudia; Anar, Burcu; Schwach, Frank; Otto, Thomas D.; Brochet, Mathieu; Volkmann, Katrin; Quail, Michael A.; Pain, Arnab ( 0000-0002-1755-2819 ) ; Rosen, Barry; Skarnes, William; Rayner, Julian C.; Billker, Oliver
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.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Computational Bioscience Research Center (CBRC)
Citation:
Pfander C, Anar B, Schwach F, Otto TD, Brochet M, et al. (2011) A scalable pipeline for highly effective genetic modification of a malaria parasite. Nature Methods 8: 1078-1082. doi:10.1038/nmeth.1742.
Publisher:
Nature Publishing Group
Journal:
Nature Methods
Issue Date:
23-Oct-2011
DOI:
10.1038/nmeth.1742
PubMed ID:
22020067
PubMed Central ID:
PMC3431185
Type:
Article
ISSN:
15487091
Appears in Collections:
Articles; Computational Bioscience Research Center (CBRC); Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorPfander, Claudiaen
dc.contributor.authorAnar, Burcuen
dc.contributor.authorSchwach, Franken
dc.contributor.authorOtto, Thomas D.en
dc.contributor.authorBrochet, Mathieuen
dc.contributor.authorVolkmann, Katrinen
dc.contributor.authorQuail, Michael A.en
dc.contributor.authorPain, Arnaben
dc.contributor.authorRosen, Barryen
dc.contributor.authorSkarnes, Williamen
dc.contributor.authorRayner, Julian C.en
dc.contributor.authorBillker, Oliveren
dc.date.accessioned2014-08-27T09:49:00Zen
dc.date.available2014-08-27T09:49:00Zen
dc.date.issued2011-10-23en
dc.identifier.citationPfander C, Anar B, Schwach F, Otto TD, Brochet M, et al. (2011) A scalable pipeline for highly effective genetic modification of a malaria parasite. Nature Methods 8: 1078-1082. doi:10.1038/nmeth.1742.en
dc.identifier.issn15487091en
dc.identifier.pmid22020067en
dc.identifier.doi10.1038/nmeth.1742en
dc.identifier.urihttp://hdl.handle.net/10754/325358en
dc.description.abstractIn 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.en
dc.language.isoenen
dc.publisherNature Publishing Groupen
dc.rightsUsers may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsen
dc.rightsArchived with thanks to Nature Methodsen
dc.subjectDNAen
dc.subjectbacteriophageen
dc.subjectbacteriophage n15en
dc.subjectcontrolled studyen
dc.subjectDNA modificationen
dc.subjectEscherichia colien
dc.subjectevolutionary homologyen
dc.subjectgenetic recombinationen
dc.subjectgenomeen
dc.subjectPlasmodium bergheien
dc.subjectshuttle vectoren
dc.subjectDNA, Protozoanen
dc.subjectDNA, Recombinanten
dc.subjectEscherichia colien
dc.subjectGene Libraryen
dc.subjectGenetic Engineeringen
dc.subjectGenetic Vectorsen
dc.subjectGenome, Protozoanen
dc.subjectHomologous Recombinationen
dc.subjectMalariaen
dc.subjectPlasmodium bergheien
dc.subjectEscherichia colien
dc.subjectPlasmodium bergheien
dc.titleA scalable pipeline for highly effective genetic modification of a malaria parasiteen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentComputational Bioscience Research Center (CBRC)en
dc.identifier.journalNature Methodsen
dc.identifier.pmcidPMC3431185en
dc.eprint.versionPost-printen
dc.contributor.institutionWellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdomen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorPain, Arnaben

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