Validation of Random Transgene Integration and Expression in Eimeria Parasites.

Validation of transgenic Eimeria parasites is essential for confirming stable genomic integration and expression of exogenous genes. This protocol describes a comprehensive workflow for verifying transgene insertion and protein expression in Eimeria tenella. The procedure begins with genomic DNA extraction from sporulated oocysts, followed by PCR amplification to preliminarily verify the presence of the target fragment. To further confirm genomic integration, whole-genome resequencing is performed to identify insertion sites and evaluate the stability of the integrated construct in the parasite genome. Protein expression is subsequently examined by Western blotting of lysates prepared from purified sporozoites, enabling detection of the target recombinant protein. In addition, intracellular localization is visualized by indirect immunofluorescence assay (IFA) using infected host cells. Together, these assays provide a robust and reproducible framework for validating transgenic Eimeria lines. This workflow enables reliable confirmation of genomic integration and protein expression, thereby supporting downstream applications in genetic manipulation, functional genomics, and vaccine development targeting Eimeria parasites.