Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect

doi:10.13560/j.cnki.biotech.bull.1985.2022-1429

Abstract

Abstract:

The aim of this study is to establish the IPEC-J2 cell line with MDH2 gene knockout by CRISPR/Cas9 genome editing technology and investigate whether MDH2 gene knockout confers resistance to deoxynivalenol. sgRNA sequences targeting MDH2 gene were designed and cloned into PX459 vector; the recombinant plasmid PX459-sgRNA-MDH2 was introduced into the IPEC-J2 cells by electroporation，and puromycin was added to screen KO cell colony generations. The MDH2 KO single cell colony generations were isolated by limited dilution method and validated by genotype sequencing, quantitative PCR and Western blotting to obtain MDH2-KO monoclonal cell colony generation. Finally, the cell viability was detected by CCK8 kit and apoptosis and necrosis assay kit to determine the resistance of MDH2 knockout cell colony generation to deoxynivalenol. The sequencing results showed that the MDH2 knockout vector was successfully constructed. The obtained cell colony generations were validated as MDH2 knockout monoclonal cell colony generations by RT-qPCR and immunoblotting. The CCK8 cell viability assay showed that knockout of MDH2 increased the viability of IPEC-J2 cells by 18.67%, 19.59%, 26.36%, and 27.01% at different concentrations of deoxynivalenol（4, 2, 1, and 0.5 μg/mL）for 5 d compared with wild-type cells. Flow cytometry assays showed that knockout of MDH2 reduced the mortality rate of IPEC-J2 cells by 30.33%, 15.81%, 16.00%, and 14.70% at different concentrations of deoxynivalenol respectively compared with wild-type cells for 5 d. We obtained the MDH2-KO monoclonal cell colony generation by editing the MDH2 gene in IPEC-J2 cells using the CRISPR/Cas9 system. The resistance of MDH2-KO cell colony generation to deoxynivalenol was demonstrated by cell viability and cell death assays, which provids insight into the toxic mechanism of deoxynivalenol-induced cell death and offers a new strategy for the prevention and treatment of deoxynivalenol.

Key words: CRISPR/Cas9 system, deoxynivalenol, MDH2 gene, cell survival, cell death

SHI Wei-tao, YAO Chun-peng, WEI Wen-Kang, WANG Lei, FANG Yuan-jie, TONG Yu-jie, MA Xiao-jiao, JIANG Wen, ZHANG Xiao-ai, SHAO Wei. Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect[J]. Biotechnology Bulletin, 2023, 39(7): 307-315.

Figures/Tables 10

Table 1 MDH2 targeting sites and sgRNA oligonucleotide sequences

引物Primer	序列 Sequence（5' -3'）
sgRNA-MDH2-F	CACCGCCCGTCATTGGCGGCCACGC
sgRNA-MDH2-R	AAACGCGTGGCCGCCAATGACGGGC

Table 2 Primers for PCR sequencing of MDH2 gene

引物Primer	序列Sequence（5'-3'）
PCR-F	CCGTCTTCAGTCCGTCGTG
PCR-R	TAACCAGCGGTGCTCCAGTG

Table 3 RT-qPCR primers for MDH2 gene

引物Primer	序列Sequence（5'-3'）
RT-qPCR-F	GGCCACGCCGGGAAG
RT-qPCR-R	GATGCCCTTTTTCCCCAGCA

Fig. 1 Sequencing peak of recombinant plasmid PX459-sgRNA-MDH2

Fig. 2 Sequencing peaks of CRISPR target sites in MDH2-KO cell line

Fig. 3 Sequencing peaks of CRISPR target sites in MDH2 wild-type IPEC-J2 cell line

Fig. 4 Sequence comparison of wild-type IPEC-J2 cells with MDH2-KO

Fig. 5 Expression assay of MDH2-KO gene A: RT-qPCR to detect the expression of MDH2. B: Western blot to identify the expressionof MDH2 protein

Fig. 6 CCK8 detects the effect of MDH2 knockdown on the anti-DON toxin effect in IPEC-J2 cells

Fig. 7 Flow assay of cell mortality of IPEC-J2 and MDH2-KO after DON treatment

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