Genetic models for post-genome functional analysis of Toxoplasma gondii

描述(申请人提供)：弓形虫是艾滋病患者的一种重要的机会性感染。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中反复出现的机会性感染。弓形虫也被越来越多地认为是一种模式的弓形虫寄生虫，这种寄生虫可能被利用来作为一个有价值的模型来研究其他相关原虫寄生虫的生物学，这些寄生虫会导致重大的人类疾病，如疟疾和隐孢子虫病(一种重要的艾滋病OI)。我们建议开发改进的遗传模型，以更有效地剖析弓形虫的基本生物学，并进一步发展这一模型。我们已经开发出了I型弓形虫菌株，由于在主要的非同源末端连接DNA修复途径中存在工程缺陷，因此同源重组的效率显著提高。这些新菌株现在能够有效和可靠地构建定向基因敲除和基因替换，以研究I型遗传背景中的基因功能。在这项建议的具体目标1中，我们将通过破坏每个菌株中的KU80基因座来培育表现出高同源重组效率的弓形虫II型和III型菌株。在具体目标2中，我们将开发一套新设计和菌株特异性遗传工具的小型试剂盒，这些工具将增强有条件地控制和遗传操作I型、III型和III型KU80基因敲除菌株的能力，这些基因敲除菌株现在表现出高频率的同源重组。这些新的菌株和遗传工具将为菌株特定毒力因子的遗传解剖提供一个基本的框架，并将通过在所有三个弓形虫谱系中进行有效的功能基因组研究，加强后基因组时代的生物发现。与公共卫生相关：该项目将开发新的遗传模型和新的弓形虫菌株，它们可以更容易地进行基因操作。更容易地对模型细胞内弓形虫病原体进行基因工程的能力，在更快地破译潜在生物学方面具有巨大的潜力，应该会导致新的治疗方法和疫苗，以对抗由寄生原虫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.