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Efficient Gene Replacements In Toxoplasma Gondii Strains Deficient For Nonhomologous End Joining, Barbara A. Fox, Jessica G. Ristuccia, Jason P. Gigley, David J. Bzik
Efficient Gene Replacements In Toxoplasma Gondii Strains Deficient For Nonhomologous End Joining, Barbara A. Fox, Jessica G. Ristuccia, Jason P. Gigley, David J. Bzik
Dartmouth Scholarship
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 approximately 500 bp were …