敲除端粒酶TERC基因的人Calu3细胞模型的构建及功能验证  

Construction and Functional Verification of human Calu3 Cell Model with Telomerase TERC Gene knockout

作　　者：张晓娜 郭鸿斌 程龙 徐山茸 金蕊 田沈[1] ZHANG Xiaona;GUO Hongbin;CHENG Long;XU Shanrong;JIN Rui;TIAN Shen(College of Life Sciences,Capital Normal University,Beijing 100048,China;The 361th Regiment Health Team of the Army Frontier Defense,Tibet 859600,China;Department of Medical Molecular Biology,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100089,China;School of Life Sciences,Anqing Normal University,Anqing 246133,China)

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]陆军边防第三六一团卫生队,西藏859600 [3]军事科学院军事医学研究院生物工程研究所,北京100089 [4]安庆师范大学生命科学学院,安庆246133

出　　处：《中国细胞生物学学报》2022年第5期848-855,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:82072717);北京市新星交叉课题(批准号:Z191100001119020)资助的课题。

摘　　要：该文基于一种可诱导的DD-Cas9系统构建敲除端粒酶TERC基因的人Calu3细胞模型,并对其功能进行验证。将靶向TERC序列的sgRNA克隆至载体pLenti-DD-Cas9-Flag上,慢病毒包装后感染Calu3细胞,对筛选获得的单克隆细胞添加小分子化合物甲氧苄啶(trimethoprim,TMP)药物诱导Cas9蛋白表达,实现对端粒酶RNA组分的敲除。单克隆细胞培养后鉴定其端粒酶活性、端粒长度的变化,并进行细胞衰老染色的实验。结果显示成功构建了pLenti-DD-Cas9-Flag-sgTERC重组质粒,并利用DD-Cas9高效、便捷的特点完成了对TERC基因的可诱导性编辑。测序结果表明,单克隆细胞中TERC序列部分敲除;TRAP实验显示端粒酶活性均有下调;提取细胞基因组用qPCR的方法进行端粒长度检测,结果显示其端粒长度明显缩短;同时,与野生型细胞相比较,TERC序列敲除的细胞出现明显衰老。总之,TERC基因的缺失导致细胞出现端粒酶活性下降、端粒长度缩短及衰老的现象,可诱导敲除端粒酶TERC基因的人Calu3细胞模型为后续对细胞衰老相关研究奠定了基础。This study aimed to construct a human Calu3 cell model with telomerase TERC gene knockout based on an inducible DD-Cas9 system,and its function was verified.The sgRNA targeting TERC sequence was cloned into the vector pLenti-DD-Cas9-Flag,and the lentivirus was packaged and infected with Calu3 cells.The monoclonal cells obtained by screening were added with the small molecule compound TMP (trimethoprim) to induce the expression of Cas9 protein to achieve the knockout of telomerase RNA components.The telomerase activity,changes in telomere length and experiments for cell senescence staining were identified after culture.The results showed that the pLenti-DD-Cas9-Flag-sgTERC recombinant plasmid was successfully constructed,and the inducible editing of the TERC gene was completed by utilizing the high efficiency and convenience of DD-Cas9.Sequencing results showed that the TERC sequence in the monoclonal cells was partially knocked out;TRAP experiment showed that the telomerase activity was down-regulated;the cell genome was extracted and the telomere length was detected by qPCR,and the results showed that the telomere length was shortened significantly.Compared with wild-type cells,TERC knockout cells showed obvious senescence.In conclusion,deletion of TERC gene leads to the phenomenon of decreased telomerase activity,shortened telomere length and senescence in cells.Human Calu3 cell model with deletion of TERC gene can be induced,which lays a foundation for subsequent studies on cell senescence.

关 键 词：CRISPR/Cas9系统 端粒酶RNA 基因敲除 细胞衰老 

分 类 号：Q343[生物学—遗传学]