利用CRISPR/Cas9慢病毒载体构建敲除大鼠肝星状细胞COX-2基因的细胞模型

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

目的通过构建CRISPR/Cas9慢病毒载体,转染HSC-T6细胞,获得能稳定表达Cas9蛋白的HSC-T6细胞和COX-2基因缺陷的HSC-T6-COX-2-/-细胞,为后期的功能研究提供良好的工具和手段,为临床上治疗肝纤维化提供新策略。方法设计合成COX-2基因特异性的sgRNA(COX-2-sgRNA-1、COX-2-sgRNA-2、COX-2-sgRNA-3),并将其连接至GV371载体上,提取重组后的质粒,将其与包装质粒共同转染到293T细胞,形成慢病毒颗粒,荧光法检测病毒滴度。按照MOI值计算病毒最适用量,先将Lenti-Cas9-puro转染至HSC-T6细胞,嘌呤霉素筛选得到HSC-T6-Cas9细胞,再用Lenti-COX-2-sgRNA-EGFP转染至HSC-T6-Cas9细胞获得HSC-T6-COX-2-/-细胞,通过Cruiser酶切检测及Western Blot等方法在基因和蛋白水平上进行敲除验证。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果通过测序验证COX-2-sgRNA表达载体构建成功。重组表达质粒和包装质粒共同转染到293T细胞,形成慢病毒颗粒,荧光法检测病毒滴度均在1×108以上。成功构建能稳定传代的Cas9蛋白的HSC-T6细胞和COX-2基因缺陷的HSC-T6-COX-2-/-细胞模型。HSC-T6-Cas9细胞中LV-Cas9-Puro mRNA相对表达量(541.93±105.76)高于CON组细胞(1.00±0.02),差异具有统计学意义(t=12.995,P<0.01)。通过Cruiser酶切检测及Western Blot试验,结果提示CRISPR/Cas9慢病毒表达载体能在靶点起作用,其中COX-2-sgRNA-2敲除作用最明显,并且COX-2蛋白表达水平较CON组和NC组相比显著下降(P值均<0.05),提示COX-2-sgRNA有活性。结论成功构建了针对COX-2靶基因的CRISPR/Cas9慢病毒载体,获得稳定的COX-2基因敲除的HSC-T6-COX-2-/-细胞。

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.