Optimization of Vectors for Stable Transfection of Acanthamoeba

描述(申请人提供)：卡氏棘阿米巴是引起眼病棘阿米巴角膜炎的几个密切相关物种之一。棘阿米巴还可以携带各种各样的细菌病原体，对这些细菌来说，它可以作为环境宿主。尽管棘阿米巴具有致病性，但它已成为广泛生化研究的模式生物，所有这些研究都因缺乏转基因系统而受阻。近年来发展了一种稳定的棘阿米巴转基因方法，为研究棘阿米巴提供了有用的工具，但第一代载体在拷贝数、阿米巴内部结构、新霉素磷酸转移酶表达水平和报告基因表达水平等方面尚未得到优化。为了建立一个在棘阿米巴中以特定水平表达蛋白质的通用系统，我们建议研究启动子强度、5‘和3’非翻译序列的性质以及内含子的存在对新霉素磷酸转移酶表达效果的影响。结果将根据质粒结构和拷贝数、转染率和新霉素磷酸转移酶产生水平进行评估。通过同源重组将产生稳定的整合子，从而建立永久细胞系，并在更生理的背景下研究基因功能。类似的方法将被用来优化同一质粒的报告基因的表达，但将包括合成的和可诱导的启动子，以便提供对表达水平的控制，范围从生理表达水平到用于重组蛋白生产的非常有效的启动子。可诱导启动子将允许有毒蛋白或针对基本蛋白的RNAi进行受控表达。串联亲和标签将被用于研究蛋白质-蛋白质相互作用和纯化低丰度多亚基复合体。总之，优化的载体将为体内研究从基因表达到致病性的过程提供一个通用的平台。卡氏棘阿米巴是一种机会性的人类病原体，可引起严重的眼病棘阿米巴角膜炎，并可引起脑部感染和免疫功能低下的人的各种感染。棘阿米巴可携带广泛的细菌和真菌病原体，这些病原体在棘阿米巴内的生长可能选择毒力。这项提议将为棘阿米巴的生物学研究以及它与其他病原体的相互作用开发基因技术。

DESCRIPTION (provided by applicant): Acanthamoeba castellanii is one of several closely related species that cause the eye disease Acanthamoeba keratitis. Acanthamoeba can also harbor a wide variety of bacterial pathogens, for which it may serve as an environmental reservoir. Notwithstanding its pathogenicity, Acanthamoeba has served as a model organism for a wide range of biochemical studies, all of which have been hampered by the lack of a transfection system. A method for stable transfection of Acanthamoeba was recently developed, providing a useful tool for several investigations, but the first generation of transfection vectors have not been optimized with respect to copy number, structure within amoebae, levels of neomycin phosphotransferase expression and reporter gene expression. In order to develop a versatile system for expressing proteins in Acanthamoeba at defined levels it is proposed to examine the effects of promoter strength, the nature of 5' and 3' untranslated sequences and the presence of introns on the efficacy of neomycin phosphotransferase expression. Results will be evaluated on the basis of plasmid structure and copy number, efficiency of transfection and levels of neomycin phosphotransferase production. Stable integrants will be produced by homologous recombination, so that permanent cell lines can be established and gene function can be studied in a more physiological context. Similar approaches will be used to optimize reporter gene expression from the same plasmid but will incorporate synthetic and inducible promoters in order to provide control over the level of expression to range from physiological expression levels to very efficient promoters for recombinant protein production. Inducible promoters will permit controlled expression of either toxic proteins or RNAi against essential proteins. Tandem affinity tags will be used in order to permit investigations of protein-protein interactions and the purification of low-abundance multi-subunit complexes. Collectively, the optimized vectors will provide a versatile platform for the in vivo study of processes ranging from gene expression to pathogenicity. Acanthamoeba castellanii is an opportunistic human pathogen causing the severe eye disease Acanthamoeba keratitis and can cause brain infection and various infections of immunocompromised individuals. Acanthamoeba can harbor a wide range of bacterial and fungal pathogens, and growth of these pathogens within Acanthamoeba may select for virulence. This proposal will develop genetic techniques for the biological study of Acanthamoeba as well as its interactions with other pathogens.