CRISPR/Cas9系统介导的猪MRC1修饰基因降低PCV2复制的研究

doi:10.11843/j.issn.0366-6964.2023.03.008

Abstract

Abstract: This study aimed to obtain target cells with MRC1 gene-modified PCV2 and verify the inhibitory effect of the cells on PCV2 in vitro, which would provide a theoretical basis for the subsequent production of gene-edited pigs.In this study:1) Firstly, a lentivirus-mediated high-efficiency homologous recombination vector and a CRISPR/Cas9 gene targeting system were established using genetic engineering technology; 2) The gene-edited MRC1 promoter in PK15 cells were screened by cell and molecular biology techniques; 3) Then, the anti-PCV2 effect was verified by PCV2 infection tests. The results showed that: 1) The pLV-LoxP-mCherry-puro-LoxP vector containing red fluorescent gene, puromycin gene, homologous LoxP sequence and multiple cloning sites was successfully constructed. The homologous arm was designed according to the differential sequences of MRC1 promoter among different pig breeds and the recombinant vector was constructed. Also the CRISPR/Cas9 mediated gene targeting vector were successfully constructed; 2) The PK15 cell line edited by MRC1 promoter gene was screened and verified by puromycin through virus packaging and co-transfection. The 14 bp upstream of the transcription start site of MRC1 gene was successfully knocked into PK15 cells; 3) The protein expression of MRC1 in PK15 cell line edited by MRC1 gene was significantly increased (P<0.05), and the replication of PCV2 was significantly reduced (P<0.05). The expression of MRC1 protein in PK15 cells edited by MRC1 promoter was significantly increased, which could be used for the preparation of anti-PCV2 pigs.

Key words: pig, CRISPR/Cas9, Cre-LoxP system, PCV2, MRC1

CLC Number:

S828.2

FEI Xiaoyu, SHI Chaoqun, LIU Xueming, SU Feng, JIANG Yunliang. CRISPR/Cas9 System Mediated Gene Modificated MRC1 in PK15 Cells Reduce PCV2 Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 934-946.

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CRISPR/Cas9 System Mediated Gene Modificated MRC1 in PK15 Cells Reduce PCV2 Replication

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