Genetic models for post-genome functional analysis of 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，以更有效地预防和治疗艾滋病复发性机会感染。刚地弓形虫也越来越被认为是一种典型的顶复合体寄生虫，这种寄生虫可能被利用作为一种有价值的模型来研究其他引起重大人类疾病的相关原生动物寄生虫的生物学，如疟疾和隐孢子虫病（一种重要的艾滋病OI）。我们建议建立改进的遗传模型，以更有效地解剖弓形虫的基本生物学，并进一步发展这种模型顶复合体寄生虫。我们已经开发出I型弓形虫菌株，由于主要的非同源末端连接DNA修复途径的工程化缺陷，其同源重组效率显著提高。这些新菌株使定向基因敲除和基因替换研究I型遗传背景下的基因功能成为可能。在本提案的具体目标1中，我们将通过破坏每种菌株的KU80位点来开发具有高同源重组效率的II型和III型弓形虫菌株。在特定目标2中，我们将开发一套小型的新工程和菌株特异性遗传工具，以增强有条件控制和遗传操作I型，II型和III型KU80敲除菌株的能力，这些菌株现在表现出高频率的同源重组。这些新菌株和遗传工具将为菌株特异性毒力因子的遗传解剖提供基本框架，并将通过对弓形虫所有三个谱系进行有效的功能基因组研究，加强后基因组时代的生物学发现。公共卫生相关性：该项目将开发新的遗传模型和新弓形虫菌株，使其更容易进行基因操纵。更容易地对细胞内病原体弓形虫模型进行基因工程改造的能力，具有更快速地破译潜在生物学的巨大潜力，并应导致新的治疗方法和疫苗，以对抗由寄生原生动物引起的人类重大疾病。

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 opportunistic infections in AIDS. Toxoplasma gondii is also being increasingly recognized as a model Apicomplexan parasite and this parasite may be harnessed to serve as a valuable model to investigate the biology of other related protozoan parasites that cause significant human diseases such as malaria and cryptosporidiosis (a significant AIDS OI). We propose to develop improved genetic models to more effectively dissect the fundamental biology of T. gondii and to further develop this model Apicomplexan parasite. We have already developed Type I strains of T. gondii that exhibit a markedly enhanced efficiency of homologous recombination due to an engineered deficiency in a major nonhomologous end-joining DNA repair pathway. These new strains now enable the efficient and reliable construction of directed gene knockouts and gene replacements to study gene function in the Type I genetic background. In specific aim 1 of this proposal we will develop Type II and Type III strains of T. gondii that exhibit a high efficiency of homologous recombination by disrupting the KU80 locus in each strain. In specific aim 2 we will develop a small kit of newly engineered and strain-specific genetic tools that will enhance the ability to conditionally control and genetically manipulate Type I, II and III KU80 knockout strains that now exhibit a high frequency of homologous recombination. These new strains and genetic tools will provide an essential framework for the genetic dissection of strain-specific virulence factors and will enhance biological discovery in the post-genome era by enabling efficient functional genomic studies in all three lineages of Toxoplasma gondii. PUBLIC HEALTH RELEVANCE: This project will develop new genetic models and novel strains of Toxoplasma gondii that can be more easily genetically manipulated. The ability to more easily genetically engineer the model intracellular pathogen Toxoplasma gondii has great potential to more rapidly decipher underlying biology and should lead to new treatments and vaccines to fight significant diseases of humans caused by parasitic protozoa.