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Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Nkrumah, LJ and Muhle, RA and Moura, PA and Ghosh, P and Hatfull, GF and Jacobs, WR and Fidock, DA (2006) Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nature Methods, 3 (8). 615 - 621. ISSN 1548-7091

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Abstract

Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB+ lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB × attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Nkrumah, LJ
Muhle, RA
Moura, PA
Ghosh, P
Hatfull, GFgfh@pitt.eduGFH
Jacobs, WR
Fidock, DA
Date: 1 August 2006
Date Type: Publication
Journal or Publication Title: Nature Methods
Volume: 3
Number: 8
Page Range: 615 - 621
DOI or Unique Handle: 10.1038/nmeth904
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
ISSN: 1548-7091
MeSH Headings: Animals; Base Sequence; Chromosomes--genetics; Genetic Engineering--methods; Integrases--genetics; Molecular Sequence Data; Mutagenesis, Site-Directed--methods; Mycobacteriophages--genetics; Plasmodium falciparum--genetics; Recombination, Genetic--genetics; Repressor Proteins--genetics; Transgenes--genetics; Viral Proteins--genetics
Other ID: NLM NIHMS234449, NLM PMC2943413
PubMed Central ID: PMC2943413
PubMed ID: 16862136
Date Deposited: 19 Nov 2012 16:56
Last Modified: 02 Feb 2019 15:57
URI: http://d-scholarship.pitt.edu/id/eprint/16375

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