摘要
目的使用CRISPR/Cas9系统靶向小鼠ATP7B基因,建立高效的ATP7B基因CRISPR/Cas9编辑系统,模拟人ATP7B基因突变,为更深入地开展威尔逊病(WD)分子机制研究和疾病干预奠定基础。方法选择我国WD的热点突变,对应小鼠ATP7B基因上的突变区域,设计单向导RNA(sgRNA),构建表达质粒,并与Cas9表达质粒共转染到小鼠N2a细胞,采用药物(嘌呤霉素和杀稻瘟菌素)筛选阳性细胞并提取gDNA,将目标位点的DNA片段进行聚合酶链式反应(PCR)扩增,然后通过TA克隆序列结果分析对目标基因的打靶效率。结果成功设计sgRNAs序列,并使用CRISPR/Cas9系统编辑了小鼠ATP7B基因,编辑效率为53.85%,创建了ATP7B基因突变细胞模型。结论通过靶向小鼠ATP7B基因,成功构建CRISPR/Cas9系统,这对深入研究WD的致病机制、探讨WD的治疗具有重要意义。
Objective To construct an efficient CRISPR/Cas9 editing system of ATP7B gene to mimic human ATP7B gene mutation,using CRISPR/Cas9 system targeting mouse ATP7B gene,so as to lay a foundation for further study of molecular mechanism and disease intervention of Wilson′s disease(WD).Methods Targeting the hot spot mutation of WD in China,the single wizard RNAs(sgRNAs)were designed,expression plasmid was constructed based on the sequence of mouse ATP7B,then the sgRNAs and Cas9 expression plasmids were co-transfected into mouse N2a cells.The positive cells were screened using puromycin and blasticidin,gDNA was extracted and the interest DNA fragments were amplified by PCR.The gene editing efficiency was evaluated by TA cloning assay.Results The sgRNAs sequence was successfully designed.Mouse ATP7B gene was edited using CRISPR/Cas9 system in N2a cells,the editing efficiency was 53.85%.ATP7B gene mutation cell model was constructed.Conclusion The CRISPR/Cas9 system is successfully constructed by targeting mouse ATP7B gene,which is of great significance for the further study of the pathogenic mechanism of WD and the treatment of WD.
作者
李欣怡
孙雪梅
付灿
岳鹏鹏
于鸿浩
李勇
LI Xinyi;SUN Xuemei;FU Can;YUE Pengpeng;YU Honghao;LI Yong(College of Biotechnology,Guilin Medical University,Guilin,Guangxi 541199,China)
出处
《现代医药卫生》
2022年第11期1801-1805,1811,共6页
Journal of Modern Medicine & Health
基金
国家自然科学基金项目(31860302)
广西壮族自治区自然科学基金项目(2018JJA140455)
广西壮族自治区科学技术厅科技计划项目(2019AC20329,2019AC20357)。