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利用CRISPR/Cas9构建G6PD基因c.1388G>A突变的HEK293/K562细胞株

文献类型: 中文期刊

作者: 张虹洋 1 ; 舒逸 1 ; 朱丹 1 ; 张佳 1 ; 邹琳 1 ; 张鹏辉 1 ;

作者机构: 1.重庆医科大学附属儿童医院临检中心;儿童发育疾病研究教育部重点实验室;国家儿童健康与疾病临床医学研究中心(重庆);儿童发育重大疾病国家国际科技合作基地;重庆市干细胞治疗工程技术研究中心

关键词: G6PD缺陷症;c.1388G>A;CRISPR/Cas9

期刊名称: 中国细胞生物学学报

ISSN: 1674-7666

年卷期: 2019 年 12 期

页码: 2263-2272

收录情况: CSCD

摘要: 该文旨在利用CRISPR/Cas9构建G6PD基因c.1388G>A突变的HEK293/K562细胞株,为G6PD缺陷症及其修复研究提供细胞模型。针对G6PD基因c.1388G>A位点设计单链向导RNA(sg RNA)与突变同源臂,利用CRISPR/Cas9联合同源重组修复(HDR)构建G6PD基因c.1388G>A突变的HEK293细胞株与红白血病K562细胞株; qRT-PCR、Western blot检测G6PD基因表达; CCK8检测细胞增殖;G6PD/6PGD比值法检测G6PD酶活性;结晶紫染色与Annexin V-APC/7-AAD验证突变细胞株对氧化活性药物维生素K3与伯安喹的耐受情况。结果显示,成功构建CRISPR/Cas9双质粒载体系统;筛选单克隆细胞经测序鉴定显示,成功构建G6PD基因c.1388G>A突变的HEK293与K562细胞株,且无脱靶;进一步发现, c.1388G>A突变不影响HEK293与K562细胞G6PD基因mRNA转录与蛋白翻译,但细胞增殖减慢, G6PD酶活性下降;突变HEK293细胞对维生素K3与伯安喹的耐受力减弱,突变K562细胞对伯安喹耐受能力减弱。该研究成功构建G6PD基因c.1388G>A突变的HEK293与K562细胞株,为G6PD缺陷症及后期基因修复研究提供细胞模型。

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