PDF(Pubmed)
Abstract：
Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of B. duncani, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of B. duncani represents an advancement over previous methods used for B. bovis [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in B. duncani, allowing for quicker and more effective selection of transgenic parasites.
摘要：
人类巴贝斯虫病是由巴贝斯虫病原体引起的蜱传疾病。疾病,表现出类似疟疾的症状，可能会危及生命,尤其是免疫系统较弱的人和老年人。世界范围内人类巴贝斯虫病的患病率逐渐上升,引起公共卫生专家的警觉。在其他病原体中，遗传技术已被证明是进行功能研究的有价值的工具，以了解特定基因在发育和发病机理中的重要性，以及验证用于药物发现的新细胞靶标。已经为几种非人类的Babesia和Theileria物种建立了遗传操作方法，最近,已经开始为人类巴贝斯虫寄生虫开发。我们先前已经报道了一种对人类病原体Babesiaduncani进行遗传操作的方法的开发。该方法基于使用hDHFR基因作为选择标记的阳性选择，其表达受ef-1aB启动子调控，以及促进通过同源重组整合到目的基因中的同源区。在这里，我们提供了在B.duncani中实施该策略以研究基因功能所需的步骤的详细描述.预期该方法的实施将显著提高我们对巴贝斯虫病的理解,并促进开发用于治疗人类巴贝斯虫病的新的和更有效的治疗策略。关键特征该方案提供了B.duncani转染的有效手段，使遗传操作和编辑，以获得对其生物学和发病机理的进一步见解。此处概述的用于B.duncani电穿孔的方案代表了先前用于B.bovis[1]的方法的进步。改进包括在电穿孔步骤期间使用的更高体积的培养物和电穿孔脉冲数的增加。这些修饰可能会提高B.duncani基因编辑的效率，允许更快，更有效地选择转基因寄生虫。
