利用CRISPR/Cas9技术构建PPARγ基因敲除的IPEC-J2细胞系

doi:10.16431/j.cnki.1671-7236.2023.07.008

Abstract

Abstract: 【Objective】 The purpose of this study was to use CRISPR/Cas9 technique to knock out the peroxisome proliferation-activated receptor γ (PPARγ) gene in porcine small intestinal epithelial cells (IPEC-J2), and to investigate the biological mechanism of PPARγ gene associated with intestinal inflammation at the cellular level.【Method】 According to porcine PPARγ gene sequence (GenBank accession No.:NM_214379), two pairs of sgRNA sequences were designed in exon 2 region.After annealing, DNA double strand was formed and connected to the eukaryotic expression vector gRNA_GFP-T1.The constructed PPARγ vector (forward target vector, reverse target vector) and Cas9-nickase plasmid were co-transfected into IPEC-J2 cells, after G418 drug screening, the monoclonal cell lines were sampled, DNA was extracted, PCR amplified, identified by gel electrophoresis and sequenced to select the cell lines with the best gene knockout effect, the expression of PPARγ protein was detected by immunofluorescence.【Result】 The target vector sequencing results showed that the target sequences were correctly connected.A total of 48 monoclonal cells were obtained after transfection and screening.The sequencing results showed that 6 monoclonal cells showed double peaks, namely KO-15, KO-17, KO-21, KO-25, KO-32 and KO-35, respectively.The KO-25 with higher basal peak was selected for immunofluorescence detection, and the results showed that the expression of PPARγ protein decreased.【Conclusion】 In this study, IPEC-J2 cells with PPARγ gene knockout were successfully obtained using CRISPR/Cas9 technology, which laid the foundation for further research on the function of PPARγ gene in inflammatory response.

Key words: CRISPR/Cas9; peroxisome proliferative activated recepter gamma (PPARγ); IPEC-J2 cells; gene knockout

CLC Number:

ZHANG Jiaxiang, HAN Diangang, SHI Yaling, MAO Xiaoyue, ZHAO Kaiwei, DU Xuan, XIN Jige. Construction of IPEC-J2 Cell Lines with PPARγ Gene Knockout Mediated by CRISPR/Cas9 Technology[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2670-2677.

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Construction of IPEC-J2 Cell Lines with PPARγ Gene Knockout Mediated by CRISPR/Cas9 Technology

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