鸡chNHE1精准基因编辑细胞系的构建及其抗ALV-J感染的研究

doi:10.11843/j.issn.0366-6964.2024.11.000

Abstract

Abstract:

This study aimed to construct a precise gene-edited cell lines for the ALV-J receptor molecule chNHE1. In DF-1 cells, key amino acids V33 and W38 facilitating ALV-J entry into host cells via chNHE1 were mutated and deleted, respectively. Meanwhile, the codon encoding amino acids 34-37 of chNHE1 were synonymously replaced. Forty-eight monoclonal cell lines were obtained through flow cytometry sorting, and PCR identification and sequencing analysis revealed that 14 of these cell lines had chNHE1 mutations in V33, W38 deletions, and synonymous substitution of codons at amino acids 34-37, with a gene editing efficiency of 29%. In order to verify the genetic stability and proliferation of the chNHE1 gene-edited cell lines, sequencing analysis was performed on the cells lines that were continuously passaged for 25 generations, and the results showed no revertant mutations in the chNHE1 gene. Further cell count analysis showed no revertant mutations in the chNHE1 gene. Further cell count analysis showed that the proliferation level of chNHE1 gene-edited cell lines was unaffected. In order to verify the anti ALV-J infection ability of the chNHE1 gene-edited cell line, ALV-J fluorescent reporter virus (ALV-J-GFP) and ALV-J prototype strain (HPRS-103) were used for virus challenge experiment. The fluorescence observation and flow cytometry analysis results showed that the chNHE1 gene-edited cell line completely resisted the infection of ALV-J fluorescent strain; Furthermore, the results of indirect immunofluorescence assays, PCR amplification assays, and virus titer determination assays showed that the chNHE1 gene-edited cell line completely resisted the infection of HPRS-103 strain. This study utilized a fluorescence labeled CRISPR/Cas9 system combined with flow cytometry sorting to successfully construct a chNHE1 gene-edited cell line, which showed complete resistance to ALV-J infection, and exhibited good genetic stability and proliferation activity. This provides theoretical support and gene editing targets for establishing new technologies against ALV-J infections.

Key words: DF-1, chNHE1, ALV-J, gene editing

CLC Number:

Xuefu ZHANG, Yuntong CHEN, Wenrui FAN, Zibo ZHANG, Mengmeng YU, Suyan WANG, Xiaole QI, Liuan LI, Yulong GAO. Construction of Chicken chNHE1 Gene Editing Cell Line and Analysis of Its Resistance to ALV-J Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5238-5246.

Figures/Tables 5

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References 21

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Construction of Chicken chNHE1 Gene Editing Cell Line and Analysis of Its Resistance to ALV-J Infection

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