Validation of Random Transgene Integration and Expression in Eimeria Parasites

Summary

Here, we present a standardized method for validating the integration and expression of foreign genes in transgenic Eimeria parasites. By integrating genomic and protein-level analyses, this workflow confirms stable gene insertion and expression. This approach facilitates the development of transgenic Eimeria for research and vaccine applications.

Abstract

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.

Introduction

Coccidiosis, caused by Eimeria species, represents one of the most economically significant parasitic diseases in poultry, leading to reduced weight gain, poor feed conversion, and global financial losses estimated in the billions of dollars each year^1,2,3. Genetic manipulation of Eimeria parasites has become a powerful tool for both fundamental research and vaccine development^4,5,6. Stable transfection enables the integration and expression of exogenous genes, such....

Protocol

All animal experiments involving chickens were approved by the Institutional Animal Care and Use Committee of China Agricultural University (approval number: CAU20160628-2). All husbandry and experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of China Agricultural University and complied with the International Guiding Principles for Biomedical Research Involving Animals. Seven-day-old male specific pathogen-free (SPF) White Leghorn chickens were used in this study. The birds were maintained under standard husbandry conditions with free access to feed and water throughout the experimental period. ....

Results

Genomic PCR analysis confirmed the presence of the target fragment in the transgenic parasites, while no corresponding bands were detected in the wild-type control (Figure 1A). Whole-genome resequencing further demonstrated that the linearized plasmid was stably integrated into the parasite genome, and the predicted integration site was validated by PCR amplification across the junction region (Figure 1B).

Protein expression was verif.......

Discussion

This protocol establishes a comprehensive workflow for validating genomic integration, protein expression, and intracellular localization of exogenous genes in transgenic E. tenella. Rather than relying on a single assay, the approach combines genomic and protein-level evidence, ensuring that transgene insertion is both structurally confirmed and functionally expressed.

Several critical steps in this protocol strongly influence the successful validation of transgenic lines. Technical .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antifade Mounting Medium	MCE	HYK1042	Fluorescent mounting medium to prevent photobleaching
BSA (3%)	Beyotime	ST023	Blocking buffer and antibody dilution buffer for IFA; block at room temperature for 30 min
Chloroform:isoamyl alcohol (24:1)	Sigma	C0549	Reagent for removing residual phenol during genomic DNA extraction
Coccidia-Free Chickens	Boehringer-Ingelheim	SPF	Used for in vivo passage and amplification of transgenic strains; approved by the Ethics Committee (approval number: CAU20160628-2)
CTAB (Cetyltrimethylammonium bromide)	Merck KGaA	H5882	Component of genomic DNA extraction buffer; formulation: 3 g CTAB + 12.27 g NaCl + 15 mL 1 mol/L Tris-HCl + 6 mL 0.5 mol/L EDTA, adjusted to a final volume of 150 mL with ddH2O
DE-52 Cellulose	Solarbio	C8350	Ion exchange chromatography medium for sporozoite purification; sequentially treated with NaOH, HCl, and distilled water, then equilibrated with glycine solution
ECL Substrate (Solution A + Solution B)	thermofisher	32106	Chemiluminescent detection reagent; mix equal volumes of Solution A and B before use
FITC-Conjugated Goat Anti-Mouse IgG	Sigma	AP124F	Fluorescent secondary antibody for IFA, dilution ratio 1:200
Glass beads	Sigma	Z250473-1PAK
Glycine Solution	Sigma	67419	Equilibration and elution buffer for chromatography columns; formulation: 0.75 g glycine + 7.9 g NaCl dissolved in 500 mL distilled water, pH adjusted to 7.4-7.6, autoclaved
Hoechst 33258	Sigma	94403	Nuclear staining reagent, dilution ratio 1:100; used for IFA localization
HRP-Conjugated Goat Anti-Mouse IgG	Sigma	12-349	Secondary antibody for Western blotting, dilution ratio 1:2000
Illumina NovaSeq Sequencing Platform	Personalbio Biotechnology Co., Ltd. (Shanghai, China)		Next-generation sequencing platform; paired-end sequencing (150 bp read length), sequencing depth 100×, generating ≥6 GB raw data (Q20>90%)
Low Speed Centrifuge	BEIJING ERA BEILI CENTRIFUGE	DT5-2
Mouse Monoclonal Anti-Flag Antibody	Sigma	F1804	Primary antibody, dilution ratio 1:200; used for IFA detection of Flag-tagged fusion protein
Mouse Monoclonal Anti-GAPDH Antibody	Sigma	G8795	Primary antibody, dilution ratio 1:2000; used as a loading control for Western blotting
Nonfat Milk (5%)	Beyotime	P0216	Blocking buffer for Western blotting; block at room temperature for 1 h or 4°C overnight
Paraformaldehyde (4%)	Merck KGaA	30525	Cell fixation solution; fix at room temperature for 30 min
PBS	Solarbio	P1010
Penicillin-Streptomycin (100×)	Yeasen	60162ES76	Antibiotic for cell culture; inhibits microbial contamination
Percoll (DG gradient stock	GE Healthcare	17-0891-09
Phenol:chloroform:isoamyl alcohol (25:24:1)	Merck KGaA	77617	Reagent for protein removal during genomic DNA extraction
Protein Loading Buffer (6×)	Beyotime	P0015F	Protein electrophoresis loading buffer; formulation: 100 mM Tris (pH 6.8), 4% SDS, 2 mM EDTA, 2% glycerol, 6 M urea; mix with 50 μL 2 M DTT and 50 μL 5% bromophenol blue before use
Proteinase K	Merck KGaA	39450-01-6	200 mg dissolved in 10 mL ddH2O, used for protein digestion during genomic DNA extraction
PVDF Membrane	thermofisher	88518	Protein transfer membrane; activated with methanol for 1 min before use
RIPA Lysis Buffer	Yeasen	20101	Lysis buffer for sporozoite protein extraction
RNase A	Merck KGaA	9001-99-4	25 mg dissolved in 2.5 mL 0.01 mol/L NaAc, heated at 100°C for 10-15 min, pH adjusted to 7.4 with 1 mol/L Tris-HCl; used for RNA removal from DNA samples
SDS-PAGE Gel Reagents (Acrylamide, Bis-acrylamide, Tris, SDS, APS, TEMED)	Bio	101	Reagents for SDS-PAGE gel preparation; acrylamide concentration adjusted according to the molecular weight of the target protein
Sodium taurodeoxycholate hydrate	Sigma	T0875
solution)
Taq Master Mix (2×)	Vazyme	P112-01/02/03	PCR amplification premix containing Taq polymerase, dNTPs, etc.
Triton X-100 (0.25%)	Merck KGaA	9036	Cell permeabilization solution; incubate at room temperature for 30 min
Trypsin	Solarbio	T8150
Vortex Mixer	Beijing North TZ-Biotech	HQ-60-II
Water Bath Thermostat	Grant Instruments (Cambridge)	GD120,GM0815010