China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (10): 4527-4537.doi: 10.16431/j.cnki.1671-7236.2025.10.001

• Biotechnology • Previous Articles    

Establishment of CRISPR/Cas9-mediated CD71 Gene Editing IPI-2I Cell Line

NIE Yuxin1, YUAN Maosha1, WANG Wanjie1, WANG Nan2, SUN Yaru1, LIU Zhiguo1, MU Yulian1   

  1. 1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China
  • Received:2025-04-10 Published:2025-09-30

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Abstract: 【Objective】 This experiment aimed to obtain cluster of differentiation 71 (CD71) gene-edited immortalized porcine intestinal-2I (IPI-2I) cell lines through CRISPR/Cas9 technology，which provided materials for further study of CD71 gene function and its role in Transmissible gastroenteritis virus (TGEV) infection at the cellular level.【Method】 Five sgRNAs were designed for the third exon and its adjacent intron region of CD71 gene,which were ligated into the pX458-GFP vector.The activity of different sgRNA vectors was detected by T7E1 enzymatic cleavage in porcine embryonic fibroblast (PEF) cells.Two sgRNA vector plasmids with higher cleavage efficiency were selected to be electrotransfected into IPI-2I cells,and after 48 h,single cells with green fluorescent protein (GFP) were collected by flow cytometry,and then cultured in 96-well cell culture plates to screen the monoclonal cells.The monoclonal cells were genotyped by PCR amplification and TA cloning to obtain the CD71 gene-edited IPI-2I cell line.【Result】 The results of plasmid sequencing showed that all five sgRNAs were successfully ligated to the pX458-GFP vector.T7E1 enzyme digestion results showed that cleavage occurred in all five transfected sgRNA plasmids.Among them,the cleavage efficiencies of pX458-sgRNA1 and pX458-sgRNA4 were relatively high,which were 35.8% and 44.1%,respectively.The results of flow cytometry sorting showed that the proportion of positive cells transfected with pX458-sgRNA1 and pX458-sgRNA4 were 29.8% and 25.7%,respectively.PCR amplification results showed that among the 65 monoclonal cells obtained,14 cells occurred gene editing,with an editing efficiency of 22%. TA cloning results showed that among the 14 gene-edited cells,5 were monoallelic edited and 9 were biallelic edited.【Conclusion】 In this study,the CD71 gene-edited IPI-2I cell line was successfully constructed using CRISPR/Cas9 technology,and CD71 gene monoallelic and biallelic gene-edited cells were obtained.The results provided a good cell model for clarifying the function of CD71 gene and its mechanism of mediating TGEV infection.

Key words: pig; CRISPR/Cas9; CD71 gene; IPI-2I cells

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