CD163单等位基因表达的iPAMs细胞系的构建及其介导PRRSV感染的特征分析

doi:10.16431/j.cnki.1671-7236.2023.07.003

Abstract

Abstract: 【Objective】 This study was to obtain an immortalized porcine alveolar macrophages (iPAMs) with CD163 (cluster of differentiation 163) monoallelic expression by using CRISPR/Cas9 gene editing technique, and explore the characteristics of their mediating Porcine reproductive and respiratory syndrome virus (PRRSV) infection, which laid the foundation for further study on the mechanism of CD163 gene in PRRSV infection.【Method】 Eight single guide RNA (sgRNA) in exon 1 of porcine CD163 gene were designed and introduced into pX458 vector, and the editing efficiency of different sgRNA vectors was detected by T7E1 assay.Three highly active sgRNA vector was electrotransfected into iPAMs, and the monoclonal cells were isolated by flow cytometry.Then genotypic identification, protein expression detection, off-target analysis and PRRSV infection analysis were performed on the obtained monoclonal cells.【Result】 The editing rate of 3 of the 8 sgRNAs was more than 28%.The results of genotype identification showed that 4 iPAMs cell lines with expected CD163 monoallelic expression were selected, with an efficiency of 8%.The results of protein expression detection and off-target analysis showed that the expression of CD163 protein significantly decreased in CD163 single allele expression cell line (P<0.05), and there was no off-target event at the predicted site.PRRSV infection analysis showed that CD163 monoallelic expression cell lines could be infected with PRRSV normally, and there was no significant difference in virus copy number and the expression of PRRSV-N protein between the CD163 monoallelic expression cells and wild-type cells (P>0.05).【Conclusion】 In this study, an iPAMs cell lines normally infecting PPRSV with CD163 monoallelic expression was constructed using CRISPR/Cas9 editing system, which laid a foundation for elucidating the role of CD163 in PRRSV infection and breeding new disease-resistant pig breeds.

Key words: CD163; Porcine reproductive and respiratory syndrome virus(PRRSV); CRISPR/Cas9; immortalized porcine alveolar macrophages (iPAMs)

CLC Number:

S828.9

WANG Hui, FENG Baoliang, XIANG Guangming, HUANG Lei, LIU Zhiguo, LI Kui, MU Yulian. Construction of iPAMs with CD163 Monoallelic Expression and Characterization of Their Mediating PRRSV Infection[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2617-2628.

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Construction of iPAMs with CD163 Monoallelic Expression and Characterization of Their Mediating PRRSV Infection

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