利用CRISPR/Cas9编辑系统构建pAPN基因敲除的IPI-2I细胞系

doi:10.16431/j.cnki.1671-7236.2021.07.002

Abstract

Abstract: The purpose of this study was to establish porcine aminopeptidase N(pAPN)gene knockout IPI-2I cell lines by CRISPR/Cas9 system,and to further investigate the role and interaction mechanism of pAPN in the process of coronavirus invasion at the cellular level.Two sgRNA vectors (pX330-GFP-g3 and pX330-RFP-g5) targeting the second exon of the pAPN gene were co-transfected into IPI-2I cells.After 48 h of transfection,the GFP and RFP fluorescent was observed in most cells,and the positive cells with the RFP and GFP fluorescent label were sorted by flow cytometry for screening monoclonal cell line.Then genomic DNA was extracted from a small number of monoclonal cells,and the editing site of pAPN gene in monoclonal cell line was identified by PCR and sequenced for obtaining the pAPN gene knockout cell line.Before and after the pAPN gene knockout,the expression of pAPN protein in the IPI-2I cells was also detected by Western blotting.The results showed that most of the IPI-2I cells could simultaneously express GFP and RFP after 48 h of transfection,indicating that pX330-GFP-g3 and pX330-RFP-g5 vectors were transferred into IPI-2I cells.PCR and sequencing results suggested that a total of 48 fragment deletion monoclonal cells were obtained (the knockout efficiency was 15.5%),of which 16 were monoallelic knockout and 32 were biallelic knockout.Among 32 strains of biallelic knockout cells,23 strains of cells were homozygous fragment knockout.Western blotting results revealed that the expression of pAPN protein could not be detected in the homozygous fragment knockout cells.In summary,this study successfully constructed a pAPN gene homozygous fragment knockout IPI-2I cell line using CRISPR/Cas9 system,which laid the foundation for elucidating the mechanism of pAPN in the process of coronavirus invasion and preparing new pig breeds that could be resistant to disease.

Key words: porcine aminopeptidase N; CRISPR/Cas9; IPI-2I cells

CLC Number:

S828.9

XU Changjiang, WANG Xiaopeng, XU Kui, ZHANG Xiuling, XIANG Guangming, ZHAO Haiquan, MU Yulian, LIN Xiao, LI Kui. Establishment of pAPN Gene Knockout IPI-2I Cell Lines Mediated by CRISPR/Cas9 System[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2282-2290.

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Establishment of pAPN Gene Knockout IPI-2I Cell Lines Mediated by CRISPR/Cas9 System

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