Journal: iScience

Article Title: Genetic manipulation for the non-model protozoan Eimeria : Advancements, challenges, and future perspective

doi: 10.1016/j.isci.2025.112060

Figure Lengend Snippet: Schematic for the development of genetically modified (GM) Eimeria oocysts The process of gene editing in Eimeria species and the subsequent evaluation of GM oocysts. Transfection and cultivation: The gene of interest (GOI), driven by a specific promoter (as detailed in Table 1 ), is delivered into the Eimeria genome via electroporation using a shuttle plasmid. This process involves the use of sporozoites, merozoites, or sporocysts as recipient cells, and may employ systems like the U-033 electroporation system. After the electroporation, the GM parasites are cultured in vitro for 24 h or more using cell lines such as Vero, MDBK, or PCKCs. The presence and expression of the reporter gene are then observed under a microscope. In vivo application : The transfected sporozoites, merozoites, or sporocysts are administered to chickens via oral, cloacal, or intravenous routes depending on the species and parasitic site of Eimeria . When drug resistance genes are present, the corresponding drug should be included in the first-generation application. It’s crucial to note that to ensure the expression of drug resistance genes in the transfected parasites, the drug is typically added 18 to 24 h after inoculation. Selection and propagation : After obtaining genetically modified oocysts, it is essential to perform reporter gene detection. Depending on the type of fluorescent reporter gene used, select the appropriate excitation wavelength for the flow cytometry channel to sort the oocysts or sporocysts. These genetically modified oocysts or sporocysts can then be enriched and subjected to further passage and amplification. When utilizing drug resistance genes, the addition of the corresponding drug can significantly enhance selection efficiency. It has been demonstrated that drug resistance genes for pyrimethamine, halofuginone, and diclazuril (unpublished data) are effective in screening GM Eimeria oocysts. Evaluation of GM oocysts : After stabilizing a population of GM oocysts, various identification methods can be employed to verify the modifications. At the genetic level, PCR can confirm the stable presence of the gene of interest, while techniques such as genome walking or plasmid rescue can analyze the genomic insertion site. Due to potential challenges in RNA extraction quality, identifying transcripts of the gene of interest using reverse transcription PCR (RT-PCR) may be technically demanding. At the protein level, GOI specific antibodies or tagged antibodies can be utilized in Western blotting (WB) and indirect immunofluorescence assays (IFA) verify the expression of GOI. In addition, depending on the purpose of genetic modification, biological analyses of GM oocysts—such as morphology, oocyst shedding patterns, pathogenicity, and immunogenicity—can be systematically compared with those of the parental strain.

Article Snippet: Lonza’s patented Nucleofector nuclear transfection technology, which integrates optimized electroporation instruments, buffers, and programs, can deliver foreign nucleic acids into the cytoplasm and directly into the nucleus, significantly improving transfection efficiency.

Techniques: Genetically Modified, Transfection, Electroporation, Plasmid Preparation, Cell Culture, In Vitro, Expressing, Microscopy, In Vivo, Selection, Flow Cytometry, Amplification, RNA Extraction, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Modification, Immunopeptidomics