CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells  

作　　者：JIANG Mao-cheng HU Zi-xuan WANG Ke-xin YANG Tian-yu LIN Miao ZHAN Kang ZHAO Guo-qi 

机构地区：[1]Institute of Animal Culture Collection and Application,College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,P.R.China [2]Faculty of Health Sciences,University of Macao,Macao 999078,P.R.China [3]Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou 225009,P.R.China [4]Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education,Yangzhou University,Yangzhou 225009,P.R.China

出　　处：《Journal of Integrative Agriculture》2023年第10期3148-3158,共11页农业科学学报（英文版）

基　　金：the National Natural Science Foundation of China(31972589);the earmarked fund for China Agriculture Research System(CARS-36);the Postgraduate Research&Practice Innovation Program of Jiangsu Province,China(KYCX21-3283)。

摘　　要：The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide(MDP)-mediated inflammatory response of bovine rumen epithelial cells(BRECs).First,changes in the m RNA expression of proinflammatory factor genes in BRECs following 10μg m L^(–1)MDP treatments were examined.RT-q PCR results showed that the expression of pro-inflammatory factor(IL-1β,IL-6,and TNF-α)m RNAs were significantly increased under MDP stimulation(P<0.001).Moreover,SLC15A4-Knockout(SLC15A4-KO)cells were obtained through lentivirus packaging,transfection,screening,and cell monoclonal culture.In order to gain further insight into the potential function of SLC15A4,we utilized transcriptome data,which revealed a change in the genes between WT-BRECs and SLC15A4-KO.Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.Meanwhile,the down-regulated genes were mostly enriched in the nuclear factorκB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways.The results of RT-q PCR also verified these detected changes.To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses,we investigated the inflammatory responses of cells exposed to MDP.WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10μg m L^(–1)MDP,in comparison to a control without MDP.Our results show that SLC15A4-KO BRECs had reduced the expression of genes(IL-6,TNF-α,CXCL2,CXCL3,CXCL9,and CCL2)and proteins(p-p65 and p-p44/42)from the MDP-mediated inflammatory response compared to WT-BRECs(P<0.05).In this experiment,CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4,and its role was confirmed via the MDP-induced inflammatory response in BRECs.This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.

关 键 词：SLC15A4 CRISPR/Cas9 immune response proton-coupled oligopeptide transporter(POT)families MDP 

分 类 号：S823[农业科学—畜牧学]