Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites

Publication date: Available online 15 January 2019Source: Molecular and Biochemical ParasitologyAuthor(s): Chunhui Duan, Dandan Hu, Xinming Tang, Jingxia Suo, Si wang, Sixin Zhang, Geru Tao, Chao Li, Chaoyue Wang, Xiaolong Gu, Xiaoli Tang, Guangping Huang, Biqi Xiang, Shaoqiang Wu, Choukri Ben Mamoun, Xun Suo, Xianyong LiuAbstractEimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocys...
Source: Molecular and Biochemical Parasitology - Category: Parasitology Source Type: research