Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout

Min PENG; Ting CAO; Xuefeng YANG; Shijie YI; Nian FU; Kebing ZHOU; Jianwu LONG

doi:10.3969/j.issn.1001-5256.2021.02.018

Volume 37 Issue 2

Mar. 2021

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Article Navigation > Journal of Clinical Hepatology > 2021 > 37(2): 336-342

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Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout

DOI: 10.3969/j.issn.1001-5256.2021.02.018

Department of Gastroenterology, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan 421002, China

Received Date: 2020-07-29
Accepted Date: 2020-10-12
Published Date: 2021-02-20

Abstract

Abstract

Objective To obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2^-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. Methods The COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2^-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Sequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of > 1×10⁸. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2^-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P < 0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P < 0.05), suggesting that COX-2-sgRNA was active. Conclusion A CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2^-/- cells with stable COX-2 gene knockout are obtained.
- Liver Cirrhosis,
- Clustered Regularly Interspaced Short Palindromic Repeats,
- Lentivirus Infections,
- Gene Knockout Techniques

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References

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Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout

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Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout

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