Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining
Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining
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DOI：
10.1128/ec.00357-08
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发表时间：
2009-04-01
期刊：
影响因子：
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通讯作者：
Bzik, David J.
中科院分区：
文献类型：
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作者：
Fox, Barbara A.;Ristuccia, Jessica G.;Bzik, David J.
高频率的非同源重组阻碍了顶端复形虫模式弓形虫的基因打靶方法。为了解决非同源末端连接（NHEJ）DNA修复途径是否可以在这种专性细胞内寄生虫中断，推定的KU蛋白进行了鉴定，并删除了预测的KU 80基因。在KU 80基因敲除中，通过在几个遗传位点的双交换同源重组的基因打靶效率被发现大于总转化体的97%。与野生型菌株相比，KU 80敲除的基因置换效率显著增加（300- 400倍）。发现仅类似于500 bp的靶DNA侧翼足以在KU 80敲除中进行有效的基因置换。KU 80基因敲除在体外稳定地保持了正常的生长速率和I型菌株的高毒力表型，但对腐草霉素或γ-辐射处理诱导的双链DNA断裂表现出增加的敏感性。总的来说，这些结果表明，一个显着的KU依赖性NHEJ DNA修复途径存在于弓形虫。整合基本上只发生在KU 80敲除背景中的同源靶向位点，使这种遗传背景成为基因靶向的有效宿主，以加速寄生虫生物学的基因组后功能分析和遗传解剖。
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 similar to 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.