Construction Of CRISPR/Cas9 Lentiviral Vector Knockout Of COX-2 Gene In Rat Hepatic Stellate Cells

Objective: By constructing the CRISPR/Cas9 lentiviral expression vector,transfection to HSC-T6 cells,obtaining HSC-T6 cells with stable expression of Cas9 protein,and HSC-T6-COX-2-/-cells with COX-2 gene defect.It provides a good tool and method for later functional research,and also provides a new strategy for clinical treatment of liver fibrosis.Methods:(1)Designed and synthesized the COX-2 gene specific sg RNA(COX-2-sg RNA-1,COX-2-sg RNA-2,COX-2-sg RNA-3),and connected it to the GV371 vector.The recombinant plasmid and the package plasmid were transfected into 293 T cells to form lentivirus particles,and the virus titer was detected by fluorescence method.(2)Calculate the most applicable value of the virus according to the MOI value.Transfection of Lenti-Cas9-puro into HSC-T6 cells,HSC-T6-Cas9 cells were screened by purinomycin,then transfected to HSC-T6-Cas9 cells by Lenti-COX-2-sg RNA-EGFP to obtain HSC-T6-COX-2-/-cells.Verification of gene and protein levels by Cruiser enzyme identification and Western blot.Results:(1)Sequencing verified that the COX-2-sg RNA expression vector was constructed successfully.(2)Recombinant expression plasmids and packaging plasmids were transfected into 293 T cells to form lentivirus particles,and the titer of the virus was more than 1E+8.(3)Successfully constructed HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/-cells with COX-2 gene defect.Detection by Cruiser enzyme and Western blot,the results suggest that the CRISPR/Cas9 lentivirus expression vector can play a role in the target,among them,COX-2-sg RNA-2 knockout was the most obvious,and COX-2 protein expression level was significantly lower than that of CON group and NC group.It is suggested that COX-2-sg RNA is active,and the difference is statistically significant(P<0.05).Conclusions:(1)Successfully constructed CRISPR/Cas9 lentivirus vector for COX-2 target gene.(2)Lenti-Cas9-puro and Lenti-COX-2-sg RNA-EGFP lentivirus vectors were successfully transfected into HSC-T6 cells,obtaining HSC-T6-Cas9 cells with stable expression of Cas9 protein,and HSC-T6-COX-2-/-cells with COX-2 gene defect,it provides an experimental basis for the study of the function and mechanism of HSC-T6 cells after COX-2 knockout.