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Figure 1 | Veterinary Research

Figure 1

From: “Self-cleaving” 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella

Figure 1

Generation of stably transfected E. tenella line co-expressing EYFP and RFP. A Schematic of the transfected vector (pSDEP2ARS). 5′ UTR (1376 bp) and 3′ UTR (1002 bp) of E. tenella surface antigen 13 (SAG13) were amplified from genomic DNA with primers SAG13-5-F/SAG13-5-R and SAG13-3-F/SAG13-3-R (Additional file 1), respectively. The synthesized 66 bp nucleotides encoding porcine teschovirus 2A sequence (Gray background and Additional file 1) was fused with the RFP gene of pMIC-EYFP/ACTss-RFP [7] by three rounds of PCR (2A-F1/2A-F2/2A-F3 and 2A-R (Additional file 1). ss: T. gondii GRA8 signal sequence. B Both EYFP and RFP were expressed in sporulated oocysts whereas no fluorescence could be detected in unsporulated oocysts. C Genomic DNA and cDNA from EtER was amplified with the primers P1/P2 (giving a 564 bp product) or P3/P4 (giving a 586 bp product) to verify the recombination of EYFP and RFP genomic DNA and cDNA from wild type E. tenella were used as a control. M: marker. D Genomic DNA from EtER was amplified with arbitrary degenerate primers (AP 1, AP 2, AP 3 and AP 4) and specific primers (5-SP 1, 5-SP 2 and 5-SP 3/3-SP1, 3-SP2 and 3-SP3 (Additional file 1) from SAG13 5′ (upper) and 3′ (lower) UTR by thermal asymmetric interlaced PCR, and the products from the third-round PCR were cloned into pEasy-T1 vector for sequencing. M: marker. E One integration site (Eth_scaff16) was confirmed by BLAST from 58 clones in E. tenella GeneDB. F Genomic DNA from EtER was amplified with the primers P5/5-SP3 (giving a 607 bp product), 3-SP3/P6 (giving a 306 bp product) and P5/P6 (giving a 6523 bp product) to verify the integration site of transfected vector in Eth_scaff16 locus in the EtER genome. Genomic DNA from wild type E. tenella was used as a control. M: marker, NS: no specific band.

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