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Eukaryotic Cell, April 2009, p. 520-529, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00357-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining{triangledown} ,{dagger}

Barbara A. Fox, Jessica G. Ristuccia, Jason P. Gigley,{ddagger} and David J. Bzik*

Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, New Hampshire 03756

Received 31 October 2008/ Accepted 2 February 2009

A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300- to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only ~500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of type I strains but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or {gamma}-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway is present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed postgenome functional analysis and genetic dissection of parasite biology.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756. Phone: (603) 650-7951. Fax: (603) 650-6223. E-mail: david.j.bzik{at}dartmouth.edu

{triangledown} Published ahead of print on 13 February 2009.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} Present address: Department of Microbiology, Immunology, and Tropical Medicine, George Washington University Medical Center, Ross Hall, 2300 I Street, Washington, DC 20037.

Eukaryotic Cell, April 2009, p. 520-529, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00357-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Huynh, M.-H., Carruthers, V. B. (2009). Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80. Eukaryot Cell 8: 530-539 [Abstract] [Full Text]  


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