State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, China.
Front Cell Infect Microbiol. 2022 Apr 6;12:844498. doi: 10.3389/fcimb.2022.844498. eCollection 2022.
Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of , a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene () and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the promoter and the reporter gene and achieved stable transfection through homologous recombination to integrate the selection marker into the parasite genome. The potential of this genetic modification method was tested by knocking out the () gene, and under the drug pressure of 5 nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for , which may facilitate gene function research and vaccine development of .
遗传修饰为剖析病原体的生物学和发病机制提供了一种非常有价值的分子工具。然而，目前尚无关于 （一种广泛存在于北美的导致人类巴贝斯虫病的病原体）遗传修饰的报道，这表明有必要开发一种遗传操作方法，以改善对原生动物病原体生物学的研究和理解策略。遗传修饰方法的建立需要启动子、选择标记和报告基因。在这里，通过常规 PCR 扩增了双拷贝基因 ()及其启动子，并通过测序进行了确认。我们利用 启动子和报告基因 建立了瞬时转染系统，并通过同源重组实现了稳定转染，将选择标记 整合到寄生虫基因组中。通过敲除 ()基因测试了这种遗传修饰方法的潜力，在药物 WR99210 的压力下，转染后第 7 天通过流式细胞术观察到 96.3%的寄生虫表达绿色荧光蛋白 (eGFP)。此外，通过有限稀释法成功获得了 敲除寄生虫的克隆系。本研究为 提供了一种转染方法，这可能有助于 的基因功能研究和疫苗开发。
