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Journal of Integrative Agriculture  2023, Vol. 22 Issue (10): 3148-3158    DOI: 10.1016/j.jia.2023.06.016
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells
JIANG Mao-cheng1, HU Zi-xuan2, WANG Ke-xin1, YANG Tian-yu1, LIN Miao1, 3, 4, ZHAN Kang1, 3, 4#, ZHAO Guo-qi1, 3, 4#
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 Macau, Macau 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
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本研究的目的是确定SLC15A4胞壁酰二肽MDP)介导的奶牛瘤胃上皮细胞(BRECs)炎症反应中的作用。首先,检测了10 μg mL-1 MDP处理后BRECs中促炎因子mRNA表达量的变化。RT-qPCR结果显示,在MDP刺激下,促炎因子(IL-1βIL-6TNF-α)的mRNA表达量显著增加(P < 0.001)。此外,通过慢病毒包装、转染、筛选和细胞单克隆培养获得了SLC15A4-KO细胞系。为了进一步了解SLC15A4的潜在功能,我们利用转录组数据揭示了野生型BRECsSLC15A4-KO之间的基因变化。鉴定出5个下调的促炎基因和13个下调的趋化因子基因与炎症反应有关。同时,下调的基因主要富集在NF-κBMAPK信号通路中。RT-qPCR的结果也证实了这些变化。为了进一步确定WTSLC15A4-KO BREC如何参与炎症反应的机制,我们研究了添加MDPBRECs的炎症反应。与对照相比,添加10 μg mL-1 MDP处理WT BRECSLC15A4-KO后,我们的研究结果表明,与野生型BRECs相比,SLC15A4-KO BRECs降低了炎症反应中(IL-6TNF-αCXCL2CXCL3CXCL9CCL2)和蛋白质(p-p65p-p44/42)的mRNA表达量(P < 0.05)。在本实验中,CRISPR-Cas9系统用于敲除奶牛瘤胃上皮细胞中的SLC15A4基因,其作用通过MDP诱导的BRECs炎症反应得到证实。这项工作将为研究MDP的促炎机制及其在奶牛亚急性瘤胃酸中毒防治工作中的应用提供理论依据。


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 mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL–1 MDP treatments were examined.  RT-qPCR results showed that the expression of pro-inflammatory factor (IL-1β, IL-6, and TNF-α) mRNAs 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 factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways.  The results of RT-qPCR 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 10 μg mL–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.

Keywords:  SLC15A4        CRISPR/Cas9        immune response        proton-coupled oligopeptide transporter (POT) families        MDP  
Received: 14 February 2023   Accepted: 21 April 2023
Fund: This study was supported by the National Natural Science Foundation of China (31972589), the earmarked fund for China Agriculture Research System (CARS-36) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX21-3283).
About author:  JIANG Mao-cheng, E-mail:; #Correspondence ZHAN Kang, E-mail:; ZHAO Guo-qi, Tel: +86-514-87997195, E-mail:

Cite this article: 

JIANG Mao-cheng, HU Zi-xuan, WANG Ke-xin, YANG Tian-yu, LIN Miao, ZHAN Kang, ZHAO Guo-qi. 2023. CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells. Journal of Integrative Agriculture, 22(10): 3148-3158.

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