Wang Sen, Wang Jianyu, Li Dongfang, Chen Fangwei, Luo Wanxin, Zhao Junlong, He Lan
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan, Hubei, China.
Bio Protoc. 2024 Jun 20;14(12):e5016. doi: 10.21769/BioProtoc.5016.
Human babesiosis is a tick-borne disease caused by 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 and 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 . 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 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 , enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of represents an advancement over previous methods used for [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 , allowing for quicker and more effective selection of transgenic parasites.
人类巴贝斯虫病是一种由病原体引起的蜱传疾病。该疾病表现出类似疟疾的症状，可能危及生命，尤其是在免疫系统较弱的个体和老年人中。人类巴贝斯虫病在全球的患病率一直在逐渐上升，这引起了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宝贵工具，有助于了解特定基因在发育和发病机制中的重要性，并验证药物发现的新细胞靶点。已经为几种非人类物种建立了遗传操作方法，最近，也开始为人类巴贝斯虫寄生虫开发此类方法。我们之前报道了一种针对人类病原体的遗传操作方法的开发。该方法基于使用hDHFR基因作为选择标记的正向选择，其表达由ef-1aB启动子调控，同时还有通过同源重组促进整合到目标基因中的同源区域。在此，我们详细描述了在[具体物种]中实施该策略以研究基因功能所需的步骤。预计该方法的实施将显著提高我们对巴贝斯虫病的理解，并促进开发治疗人类巴贝斯虫病的新型且更有效的治疗策略。关键特性 本方案提供了一种有效的[具体物种]转染方法，能够进行遗传操作和编辑，以进一步深入了解其生物学和发病机制。此处概述的[具体物种]电穿孔方案代表了对先前用于[具体物种]的方法的改进[1]。改进之处包括在电穿孔步骤中使用更高体积的培养物以及增加电穿孔脉冲的数量。这些修改可能提高[具体物种]中基因编辑的效率，从而能够更快、更有效地选择转基因寄生虫。
