Enhanced homologous recombination in Toxoplasma gondii

描述（申请人提供）：弓形虫是艾滋病患者的重要机会性感染。迫切需要改进战略和方法，以更有效地预防和治疗艾滋病期间的复发性感染。由于体外培养和克隆的容易性、转化的效率、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顶复门寄生虫的模型。T.刚地也被用作替代寄生虫模型，以研究隐孢子虫（Cryptosporidiumparvum）的生物学，隐孢子虫是艾滋病流行背景下的另一种非常重要的机会致病菌，以及其他重要的顶复门寄生虫，如疟原虫和艾美耳球虫。为了更有效地利用T.弓形虫作为其他寄生虫的替代模式生物以及加速弓形虫病的改进的预防和治疗策略的发展，我们建议开发具有增强的同源重组效率的遗传改良的模式生物弓形虫。像T型车。弓形虫可用于改进异源表达弓形虫基因的模型系统。细小病毒或其他病原体，并开发基因确定的疫苗株。T.具有增强的同源重组的弓形虫菌株可以潜在地加速功能基因组学，以更有效地识别和验证新的药物靶点，以及增强弓形虫后基因组时代的生物发现方面。刚地。构建这些新的T.弓形虫，我们建议开发遗传定义的寄生虫菌株，是缺乏非同源末端连接重组修复的基础上，在T。与KU70和KU80对应的弓形虫基因同源物。我们预测KU70和KU80在T.弓形虫感染将导致同源重组显著增强的表型。这些研究将开发和验证菌株，可能是有用的模式生物更有效地构建定义的基因敲除和基因操作的弓形虫。

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.