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复发性机会感染。刚地弓形虫也越来越被认为是一种顶复门寄生虫模型，这种寄生虫可能被利用来作为一种有价值的模型，以研究其他相关的原生动物寄生虫的生物学，这些寄生虫会引起重大的人类疾病，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T。弓形虫，并进一步发展这种模式Apicomplexan寄生虫。我们已经开发出T.由于主要非同源末端连接DNA修复途径中的工程缺陷而表现出显著增强的同源重组效率的弓形虫。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置换，以研究I型遗传背景中的基因功能。在本提案的具体目标1中，我们将开发T的II型和III型菌株。通过破坏每个菌株中的KU80基因座而表现出高同源重组效率的弓形虫。在具体目标2中，我们将开发一个新工程化的和菌株特异性遗传工具的小试剂盒，其将增强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。这些新的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必要的框架，并将通过在所有三个谱系的弓形虫中进行有效的功能基因组研究来增强后基因组时代的生物发现。公共卫生相关性：该项目将开发新的基因模型和新的弓形虫菌株，这些基因模型和菌株可以更容易地进行基因操作。更容易地对模型细胞内病原体弓形虫进行基因工程的能力具有更快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.