Enhanced homologous recombination in Toxoplasma gondii

描述（申请人提供）：刚地弓形虫是艾滋病患者重要的机会性感染。迫切需要改进战略和方法，以便更有效地预防和治疗艾滋病期间的复发感染。由于体外培养和克隆的容易性、转化的效率、已建立的遗传工具的丰富性以及小鼠感染模型的可用性，弓形虫越来越被认为是一种典型的顶复合体寄生虫。刚地弓形虫也被用来作为替代寄生虫模型来研究小隐孢子虫（隐孢子虫是艾滋病大流行背景下另一种非常重要的机会性病原体）和其他重要的顶复体寄生虫，如疟原虫和艾美耳虫。为了更有效地利用弓形虫作为其他寄生虫的替代模式生物，加快弓形虫病预防和治疗策略的发展，我们提出开发具有更高同源重组效率的遗传改良弓形虫模式生物。这种模型弓形虫寄生虫可用于改进模型系统，用于小弓形虫或其他病原体基因的异源表达，并开发遗传上确定的疫苗菌株。弓形虫同源重组菌株的增强可能会加速功能基因组学的发展，从而更有效地识别和验证新的药物靶点，并促进弓形虫后基因组时代的生物学发现。为了构建这些新的弓形虫寄生虫，我们建议在构建弓形虫KU70和KU80基因同源物的明确敲除基因的基础上，开发缺乏非同源末端连接重组修复的遗传确定的弓形虫菌株。我们预测KU70和KU80在弓形虫中的敲除将导致表型显着增强的同源重组。这些研究将开发和验证可能成为有用的模式生物的菌株，用于更有效地构建刚地弓形虫的定义基因敲除和基因操作。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an important opportunistic infection of AIDS patients. Improved strategies and approaches are urgently needed to more effectively prevent and treat recurrent infections during AIDS. Due to the ease of in vitro culture and cloning, efficiency of transformation, abundance of established genetic tools, and availability of murine models of infection, Toxoplasma gondii is increasingly recognized as a model apicomplexan parasite. T. gondii is also being adopted as a surrogate parasite model to investigate biology of Cryptosporidium parvum, another very important opportunistic pathogen in the context of the AIDS pandemic, and other important apicomplexan parasites such as Plasmodium and Eimeria. To more effectively utilize T. gondii as a surrogate model organism for other parasites as well as to accelerate the development of improved prevention and treatment strategies for Toxoplasmosis, we propose to develop genetically improved model organism T gondii parasites with an enhanced efficiency of homologous recombination. Such model T. gondii parasites could be used to improve model systems for heterologous expression of genes from C. parvum or other pathogens and to develop genetically defined vaccine strains. T. gondii strains with enhanced homologous recombination can potentially accelerate functional genomics to more effectively identify and validate new drug targets, as well as to enhance aspects of biological discovery in the post-genomic era of T. gondii. To construct these novel T. gondii parasites, we propose to develop genetically defined parasite strains that are deficient in nonhomologous end-joining recombination repair based on the genetic construction of defined knockouts in the T. gondii gene homologs corresponding to KU70 and KU80. We predict that KU70 as well as KU80 knockouts in T. gondii will result in a phenotype of significantly enhanced homologous recombination. These studies will develop and validate strains that are likely to be useful model organisms for the more efficient construction of defined gene knockouts and gene manipulations in Toxoplasma gondii.