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目的:利用CRISPR/Cas9系统在HEK293FT细胞系中敲除CYP2E1基因并探讨其在检测化学物毒性中的应用。方法:设计3对分别靶向CYP2E1基因第2、第3和第7外显子的sg RNA序列并构建PX461表达载体;将携带靶向CYP2E1的sg RNA表达载体转染至HEK293FT细胞中,通过SURVEYOR检测分析sg RNA的切割效率;利用有限稀释法获得CYP2E1敲除细胞株,通过实时荧光定量PCR和蛋白印迹检测细胞株中CYP2E1的敲除效果;并检测其对N-(4-羟基苯基)乙酰胺和1,2-二氯乙烷的细胞毒性效应的影响。结果:SURVEYOR检测分析靶向CYP2E1基因第3外显子的sg RNA的切割效率为23%;成功构建了2株CYP2E1基因敲除的细胞株,实时荧光定量PCR结果显示CYP2E1 m RNA表达下降,蛋白印迹结果显示CYP2E1蛋白无表达;CYP2E1基因敲除会显著降低N-(4-羟基苯基)乙酰胺和1,2-二氯乙烷的细胞毒性。结论:通过CRISPR/Cas9构建出CYP2E1稳定敲除的肾细胞系,为研究CYP2E1的功能和作用机制提供了有效的工具。
OBJECTIVE: To knock out CYP2E1 gene in HEK293FT cell line using CRISPR / Cas9 system and to explore its application in the detection of chemical toxicity. Methods: Three pairs of sg RNAs targeting at the second, third and seventh exons of CYP2E1 gene were designed and constructed, respectively. The sg RNA expression vector carrying CYP2E1 was transfected into HEK293FT cells, Detection and analysis of sg RNA cleavage efficiency; obtained by limited dilution CYP2E1 knockout cell lines, by real-time fluorescent quantitative PCR and Western blot detection of cell lines CYP2E1 knockout effect; and test its N- (4-hydroxyphenyl) Acetamide and 1,2-dichloroethane. Results: SURVEYOR assay showed that the efficiency of sg RNA targeting to exon 3 of CYP2E1 gene was 23%. Two CYP2E1 knockout cell lines were constructed successfully. The results of real-time fluorescence quantitative PCR showed that the expression of CYP2E1 mRNA decreased. The results of western blotting showed no expression of CYP2E1 protein; CYP2E1 knockdown significantly reduced the cytotoxicity of N- (4-hydroxyphenyl) acetamide and 1,2-dichloroethane. CONCLUSION: CYP2E1 stable knockout renal cell line was constructed by CRISPR / Cas9, which provided an effective tool for studying the function and mechanism of CYP2E1.