基于CRISPR/Cas9系统建立DNA结合抑制因子3基因敲除人皮肤成纤维细胞系

Construction of inhibitor of DNA binding 3 gene knockout human skin fibroblast cell line based on CRISPR/Cas9 system

目的利用CRISPR/Cas9技术建立转录因子DNA结合抑制因子3(ID3)稳定敲除的人皮肤成纤维细胞系(BJ-5ta),为研究ID3生物学功能提供细胞模型。方法针对ID3基因的生物学特性设计3对小向导RNA(sgRNA),将其分别插入LentiCRISPR v2载体中,构建重组质粒;用构建的慢病毒载体感染BJ-5ta细胞,通过嘌呤霉素筛选获得ID3基因稳定敲除的BJ-5ta细胞系;利用T7核酸内切酶Ⅰ酶切实验和Western印迹实验检测ID3基因敲除效果;利用逆转录实时定量PCR(RT-qPCR)实验检测ID3下游负调控基因转录产物的表达情况;利用CCK-8试剂盒检测敲除ID3基因对BJ-5ta细胞增殖的影响。结果T7核酸内切酶Ⅰ酶切和Western印迹实验结果表明利用CRISPR/Cas9技术有效敲除BJ-5ta细胞ID3基因;RT-qPCR结果表明ID3基因敲除后其下游负调控基因转录产物表达上调;CCK-8实验结果表明ID3基因敲除对BJ-5ta细胞增殖无影响。结论成功构建人源ID3基因敲除BJ-5ta细胞系,可为研究ID3调控机制和深入了解其在人皮肤成纤维细胞中的生物学功能提供细胞模型。

Objective To construct a human skin fibroblast cell line(BJ-5ta)with stable knockout of inhibitor of DNA binding 3(ID3)genes using the CRISPR/Cas9 technique in order to provide a cell model for elucidating the biological function of ID3.Methods In line with the biological characteristics of ID3 genes,three pairs of small guide RNA(sgRNA)were designed and inserted into LentiCRISPR v2 vector to construct recombinant plasmids.BJ-5ta cells were infected with the constructed lentiviral vector,and ID3-knockout BJ-5ta cell lines were selected through puromycin.The efficiency of ID3 gene knockout was detected by T7 endonucleaseⅠdigestion and Western blotting.The expression levels of downstream negative regulatory gene transcription products were detected via reverse transcription real-time quantitative PCR(RT-qPCR).The effect of ID3 gene knockout on cell proliferation in BJ-5ta cells was detected using CCK-8 kit.Results T7 endonucleaseⅠdigestion and Western blotting results showed that ID3 genes in BJ-5ta cells were effectively knocked out by the CRISPR/Cas9 system.RT-qPCR results showed that the expressions of downstream negative regulatory gene transcripts were up-regulated by ID3 gene knockout.The results of CCK-8 assay showed that ID3 gene knockout had no effect on the proliferation of BJ-5ta cells.Conclusion Human BJ-5ta cell lines with ID3 knockout have been constructed,which can provide a cell model for studying the regulatory mechanism of ID3 and for gaining insights into the biological function of ID3 in human skin fibroblast cell lines.