Optimization of Vectors for Stable Transfection of Acanthamoeba

描述（申请人提供）：castellanacanthamoeba castellanii是引起眼病棘阿米巴角膜炎的几个近缘物种之一。棘阿米巴也可以容纳各种各样的细菌病原体，它可能作为一个环境水库。尽管棘阿米巴具有致病性，但它已被广泛用作生化研究的模式生物，所有这些研究都因缺乏转染系统而受到阻碍。一种稳定转染棘阿米巴的方法最近被开发出来，为一些研究提供了有用的工具，但第一代转染载体在拷贝数、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方面还没有得到优化。为了开发一个在棘阿米巴中表达蛋白的通用系统，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、转染效率和新霉素磷酸转移酶的产生水平进行评估。稳定的整合体将通过同源重组产生，从而可以建立永久细胞系，并可以在更生理的背景下研究基因功能。类似的方法将用于优化来自同一质粒的报告基因表达，但将纳入合成和诱导启动子，以便控制从生理表达水平到重组蛋白生产的非常有效的启动子的表达水平。诱导启动子将允许毒性蛋白或RNAi对必需蛋白的控制表达。串联亲和标签将用于蛋白质-蛋白质相互作用的研究和低丰度多亚基复合物的纯化。总的来说，优化的载体将为从基因表达到致病性等过程的体内研究提供一个通用的平台。castellanacanthamoeba castellanii是一种导致严重眼病棘阿米巴角膜炎的机会性人类病原体，可引起脑感染和免疫功能低下个体的各种感染。棘阿米巴可以容纳多种细菌和真菌病原体，这些病原体在棘阿米巴体内的生长可能会选择毒性。这一建议将为棘阿米巴及其与其他病原体的相互作用的生物学研究开发遗传技术。

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.