利用CRISPR/Cas9系统构建LMNA基因突变 AC16人心肌细胞系  

Construction of AC16 Human Cardiomyocyte Cell Line with Mutant LMNA Gene by CRISPR/Cas9

作　　者：邹镭 邢兵 杨柳[1,2] ZOU Lei;XING Bing;YANG Liu(College of Life Science,Guangxi Normal University,Guilin Guangxi 541006,China;Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection(Guangxi Normal University),Ministry of Education,Guilin Guangxi 541006,China)

机构地区：[1]广西师范大学生命科学学院,广西桂林541006 [2]珍稀濒危动植物生态与环境保护教育部重点实验室(广西师范大学),广西桂林541006

出　　处：《广西师范大学学报（自然科学版）》2023年第3期163-170,共8页Journal of Guangxi Normal University:Natural Science Edition

基　　金：国家自然科学基金(31560319)。

摘　　要：LMNA基因突变引发核纤层蛋白(LaminA/C)及其互作蛋白表达异常,引起机体一系列生理学反应,导致核纤层蛋白病发生,目前其具体致病机制尚不清楚。本文利用CRISPR/Cas9技术,选取符合sgRNA筛选原则的致病突变位点,成功构建了携带LMNA突变Q517X的细胞系,并对突变细胞系与LaminA/C相连或互作的LaminB1、PCNA、P53、PKCα等基因的RNA表达水平与蛋白表达水平及突变细胞核膜骨架形态进行检测,发现:Q517X突变细胞的LaminB1、P53、PCNA基因mRNA表达量显著降低约70%;LaminA/C蛋白几乎不表达,PKCα、LaminB1、P53等蛋白的表达量分别降低75%、60%、80%。结果表明,Q517X突变通过改变AC16人心肌细胞LMNA相关基因的表达进而影响细胞核正常骨架结构与功能。Mutations in LMNA(LaminA/C)gene trigger abnormal expression of nucleofibrillar proteins and their linked or interacting proteins,causing a series of physiological responses in the body and leading to the development of nucleofibrillar protein disease,however,the specific pathogenic mechanism of the disease is still unclear.In this paper,a cell line carrying LMNA mutation Q517X is successfully constructed by using CRISPR/Cas9 technology to select pathogenic mutation loci in accordance with the sgRNA screening principle,and examined the RNA expression levels and protein expression levels of LaminB1,PCNA,P53,PKCαand other genes linked to or interacting with LaminA/C in the mutant cell line and the nuclear membrane of the mutant cells.It was found that Q517X mutant cells were not mutated,and the mRNA expression of LaminB1,P53 and PCNA genes were significantly reduced by about 70%in Q517X mutant cells,and the expression of LaminA/C,PKCα,LaminB1 and P53 proteins were significantly reduced by 75%,60%and 80%respectively in Q517X mutant cells.The results showed that the Q517X mutation altered the expression of LMNA-related genes in human cardiomyocytes and thus affected the normal cytoskeletal structure and function of the nucleus.

关 键 词：LMNA 核纤层蛋白 核纤层蛋白病 CRISPR/Cas9 基因编辑 

分 类 号：Q78[生物学—分子生物学] R542.2[医药卫生—心血管疾病]