Establishment of CRISPR/Cas9-mediated HO-1 knockout cell line andits applica-tions
10.16303/j.cnki.1005-4545.2024.11.21
- VernacularTitle:CRISPR/Cas9介导的HO-1基因敲除细胞系的建立及其应用
- Author:
Shengzhe SONG
1
;
Tongwang LUO
;
Lingjun SHEN
;
Shujie WANG
;
Xiaoqiang YU
;
Houhui SONG
;
Chunyan SHAO
Author Information
1. 浙江农林大学动物科技学院/动物医学院,浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程实验室/动物医学与健康管理浙江省国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江 杭州 311300
- Keywords:
HO-1;
PK-15;
gene knockout;
CRISPR/Cas9;
cadmium
- From:
Chinese Journal of Veterinary Science
2024;44(11):2463-2469
- CountryChina
- Language:Chinese
-
Abstract:
Porcine kidney cell line(PK-15)with HO-1 gene knockout was constructed by CRISPR/Cas9 system to provide experimental materials for exploring the role and molecular mechanism of HO-1 in cadmium-induced ferroptosis in PK-15 cells.Three sgRNA targeting HO-1 gene were de-signed,synthesized and ligated to lentiviral vector Lenti CRISPRv2.The constructed lentiviral plas-mid was transfected into human embryonic cells(HEK293FT)to obtain recombinant lentivirus,which was used to infect PK-15 cells.The monoclonal cell line with HO-1 knockout was obtained by puromycin screening and limited dilution method.The knockout effect was analyzed by sequencing and Western blot detection.The HO-1 gene knockout cells were treated with 10 μmol/L cadmium chloride(CdCl2).The cell viability was detected by CCK-8 method,the cell morpholo-gy was observed by phase contrast microscope,and the content of ferrous ion(Fe2+)was detected by fluorescence probe.The results showed that sgRNA2 possessed the highest editing efficiency.The base insertion or deletion of HO-1 gene and the frameshift of the target gene occurred in all five knockout cells.Western blot results showed that no expression of HO-1 protein was detected,indicating that the PK-15 cell line with HO-1 gene knockout was successfully constructed.After CdCl2 treatment,compared with the control cells,the cell viability was significantly increased and the Fe2+content was significantly decreased in PK-15 cells with HO-1 gene deletion,indicating that HO-1 gene knockout could significantly alleviate the abnormal iron metabolism caused by cadmium treatment.In conclusion,this study successfully constructed a PK-15 cell line knocking out HO-1 gene by using CRISPR/Cas9 gene editing technique,and confirmed that HO-1 plays an important role in iron metabolism abnormality in PK-15 cells induced by cadmium treatment,which lays a foundation for further study on the regulatory mechanism of HO-1 in ferroptosis in PK-15 cells induced by cadmium exposure.
