Establishment of a porcine small intestinal epithelial cell line with IRF8 gene knockout based on AAV-SaCas9
10.16303/j.cnki.1005-4545.2025.06.09
- VernacularTitle:基于AAV-SaCas9的猪小肠上皮细胞IRF8基因敲除细胞系的建立
- Author:
Mingliang ZHANG
1
;
Kaiqi LIAN
;
Yao WANG
;
Bingqian WANG
;
Shengming MA
;
Yifan ZHANG
;
Xinying JI
;
Xuekun DOU
;
Longfei ZHANG
;
Shaoting WENG
Author Information
1. 安阳工学院生物与食品工程学院,河南安阳 455000
- Publication Type:Journal Article
- Keywords:
porcine intestinal epithelial cells;
interferon regulatory factor 8;
CRISPR/Cas9;
gene knockout
- From:
Chinese Journal of Veterinary Science
2025;45(6):1169-1177
- CountryChina
- Language:Chinese
-
Abstract:
The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P＜0.05)and IFN-λ(P＜0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.
