Transgenic Eimeria Genome Integration Verification

To confirm the integration of the exogenic DNA fragment into the genome of transgenic E. acervulina, the flanking sequences of the 5′ integration site were identified using a genome walking kit (Takara, Dalian, China). The extraction and validation of genomic DNA from transgenic E. acervulina was performed according to the previously described methods [9 (link)]. Specific primers (SP) were designed according to E. tenella Sag13 promoter sequence as previously described [17 (link)]. PCR products of the third round were recovered and cloned into the pEASY-T1-simple vector (TransGen Biotech, Beijing, China). The sequencing results were analyzed by DNAStar7.0 software, and the integration sites in the genome were identified by performing a BLAST search in the E. acervulina DB database [22 (link)].