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| Targeted Knockout of ace2 in Porcine Intestinal Epithelial Cells (IPEC-J2) by CRISPR/Cas9 System and Its Functional Analysis |
| WANG Kai, WANG Huan-Huan, LIU Ying, JI Xiao-Xia, ZHANG Yuan-Shu* |
| Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract Studies about angiotensin converting enzyme 2 (ACE2) have focused on rodents and humans (Homo sapiens), and there are few data on pigs (Sus scrofa domesticus). The good anti-inflammatory and anti-injury effects of ACE2 in different tissues and organs have been widely recognized, but the role of ACE2 in intestinal tract is still unclear. This study intends to use the CRISPR/Cas9 gene editing technology to establish ace2 gene knockout method in porcine intestinal epithelial cell (IPEC-J2), and to explore the effect of ace2 deletion on cellular inflammatory injury. First, the presence of ACE2 protein in selected IPEC-J2 cells was confirmed by Western blot and immunofluorescence staining. The ace2 gene was then selected as the knockout target, and 3 pairs of single guide RNA (sg RNA) were designed and synthesized, and inserted into the pX330 plasmid containing Cas9 backbone to construct the ace2 knockout targeting vector pX330-ace2-1, pX330-ace2-2 and pX330-ace2-3. The correct recombinant plasmid pX330-ace2-2 was transfected into IPEC-J2 cells. Western blot and immunofluorescence staining showed that ACE2 protein expression was absent. The inflammatory factors were detected by enzyme linked immunosorbent assay (ELISA). The results showed that lipopolysaccharide (LPS) treated ace2 knockout cells and the pro-inflammatory factors interleukin (IL)-1β and IL-8 were significantly increased (P<0.01), the anti-inflammatory factor IL-10 was significantly decreased (P<0.05), and the cellular inflammatory injury was aggravated which suggested that intestinal ACE2 might had certain anti-inflammatory effect. The present study established a method for knocking out ace2 gene in pig intestinal epithelial cells by CRISPR/Cas9 system, and preliminarily explored the anti-inflammatory effect of ACE2, which might provide basic information for further research on ACE2 related functions and mechanisms.
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Received: 11 September 2018
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Corresponding Authors:
zhangyuanshu@njau.edu.cn
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