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| Construction of Gene Targeting Vectors for MUC4 and SLC12A8 in Pig(Sus scrofa) by CRISPR/Cas9 System |
| WANG Wen-Wen1, YU Ying2, ZHANG Qin1,2,* |
1 College of Animal Science and Technology, Shandong Agricultural University, Tai'an 271018, China;
2 Key Laboratory of Agricultural Animal Genetics and Breeding, Ministry of Agriculture/National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China |
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Abstract Enterotoxigenic Escherichia coli (ETEC) F4ac is a major pathogenic bacteria causing diarrhea in newborn piglets (Sus scrofa). MUC4 and SLC12A8 influence the susceptibility of piglets to ETEC F4ac, and these 2 genes are located on the loci controlling F4ac receptor towards QTL mapping. In this study, 5 MUC4 and 6 SLC12A8 gene knockout vectors were constructed through CRISPR/CAS9 system. First, 5 and 6 single guide RNAs (sgRNAs) targeting to porcine MUC4 and SLC12A8 genes were designed and inserted into the pX330 plasmid. There were 3 and 5 knockout vectors, separately, which were verified efficiently using the T7E1 enzyme digestion assay after transfected into IPEC-J2 cells. The results of T7E1 enzyme digestion assay showed that the mutant efficiency for 3 knockout vectors of MUC4 amounted to 8.4%, 6.7% and 6.2%. The mutant efficiency for 5 SLC12A8 knockout vectors amounted to 18.8%, 19.1%, 12.2%, 18.1% and 13.1%, respectively. Then Western blot analyses were performed to further detect the expression of targeted genes after transfection. The results showed that the expressions of MUC4 were downregulated with varying degrees after transfection with pX330-MUC4-3, pX330-MUC4-4 and pX330-MUC4-5, and pX330-MUC4-4 vector had the highest knockout efficiency. Among the 5 target vectors of SLC12A8, transfection with pX330-SLC12A8-2, pX330-SLC12A8-5 and pX330-SLC12A8-6 downregulated the expressions of SLC12A8 with varying degrees, and pX330-SLC12A8-5 vector had the highest knockout efficiency. This study could provide experimental materials and technical support for cultivation of MUC4 and SLC12A8 gene knockout cell lines, and contribute important materials for understanding of the gene function in the future.
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Received: 06 December 2019
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Corresponding Authors:
* qzhang@sdau.edu.cn
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