摘要
CRISPR/Cas9是新一代基因组编辑技术,可简便快捷地在哺乳动物细胞对基因进行敲除、敲入。但常规的CRISPR/Cas9表达系统直接转染效果差、病毒包装效率低,极大地限制了CRISPR/Cas9系统的广泛使用。该研究应用Tet-on系统,建立了Dox诱导Cas9表达的293T细胞株,命名为293T-i Cas9。MEIS1(myeloid ectropic viral integration site 1)是TALE(three amino acid loop extension)同源域家族的转录因子,其在白血病发生发展、胚胎造血系统发育及神经系统发育中有重要作用,但其作用机制仍未完全明确。将靶向MEIS1 Exon3的sg MEIS1表达载体转入293T-iCas9,SURVEYOR实验和Western blot检测结果表明,sg MEIS1有效地指导Cas9进行基因组编辑。最终经测序和Western blot结果证明,成功建立了MEIS1敲除细胞株,这为研究MEIS1的功能提供了重要的工具。
CRISPR/Cas9 is a novel genome editing tool which enables rapid and highly efficient generation of biallelic knockout cell lines for loss-of-function studies, as well as homozygous knockin cell lines with specific nucleotide alterations for disease modeling. However, there are lots of limitations in common CRISPR/Cas9 system, such as low efficiency of direct transfection and virus packaging. In this study, we used a Tet-on system to establish an inducible genome-engineering platform which can express the Cas9 with Doxychline treatment in HEK293T cell line named as 293T-iCas9 cell line. MEIS 1, a transcription factor of TALE homeodomain family, plays important roles in leukemia, hematopoiesis and the nervous development. However, the mechanism by which MEIS 1 regulates these processes remains unclear. We transfected a sgRNA expression vector targeting the Exon3 of MEIS 1 into 293T-iCas9 cell line. SURVEVOR assay and Western blot results demonstrated that sgMEIS 1 was able to edit genomic DNA at MEIS1 loci. MEIS1 knockout cell line had been derived successfully after further confirmation by sequecing and Western blot. It could be used as an important tool for futher study of the function of MEIS1.
作者
张明智
王梦鸽
王洪涛
苏培
刘鑫
张磊升
刘翠翠
王钰
吴丹
周家喜
彭莎
Zhang Mingzhi Wang Mengge Wang Hongtao Su Pei Liu Xin Zhang Leisheng Liu Cuicui Wang Yu Wu Dan Zhou Jiaxi Peng Sha(College of Veterinary Medicine, Shaanxi Stem Cell Engineering and Technology Research Center, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest Agriculture and Forestry University, Yangling 712100, China Stata Key Laboratory of Experimental Hematology, Institute of Hematology ＆ Blood Diseases Hospital, Chinese Academy of Medical Sciences ＆ Peking Union Medical College, Tianjin 300020, China)
出处
《中国细胞生物学学报》
CAS
CSCD
2016年第8期978-985,共8页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:31500949)
国家重大科学研究计划(批准号:2012CB966403、2015CB964902)
北京协和医学院“协和青年基金”(批准号:3332015128)
教育部留学回国人员启动经费(批准号:K308021501)
留学人员科技活动项目择优资助经费(批准号:A289021502)
西北农林科技大学基本科研业务费(批准号:2452015034)资助的课题~~
