利用CRISPR/Cas9技术构建22q-Enh3增强子元件敲除型A549稳定细胞系  

Construction of 22q-Enh3 Enhancer Knockout A549 Cell Line by CRISPR/Cas9 System

作　　者：俞大伟[1,2] 杨音[1,2] 邵立培 孙鸾[1,2] 杨楠[1,3] 孙玉洁[1,2] 

机构地区：[1]南京医科大学江苏省人类功能基因组学重点实验室,南京市210029 [2]南京医科大学细胞生物学系,南京市210029 [3]南京医科大学生物化学与分子生物学系,南京市210029

出　　处：《医学分子生物学杂志》2017年第2期63-67,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81572789）

摘　　要：目的 增强子元件敲除细胞系是探索增强子功能的理想细胞模型,为了探索位于22q12.2肺癌易感染色质区的增强子元件22q-Enh3的生物学功能,建立敲除22q-Enh3增强子元件的纯合细胞系.方法 利用CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/crispr-associated 9）基因敲除技术在非小细胞肺癌A549细胞系中敲除22q-Enh3增强子,并运用流式细胞术、细胞培养以及PCR技术筛选和鉴定敲除型克隆.结果 我们最终获得了3个敲除增强子元件22q-Enh3的纯合子细胞克隆.结论 本研究为进一步研究22q-Enh3增强子元件的生物学功能提供了细胞模型,并为应用CRISPR/Cas9基因敲除技术建立其他增强子元件敲除型纯合子细胞模型积累了宝贵经验.Objective The enhancer knockout cell line is an ideal model to investigate the enhancer function.This study aimed to construct the homozygous 22q-Enh3 knock out cell line in an attempt to investigate the biological function of 22q-Enh3 located in lung cancer risk region 22q12.2.Methods 22q-Enh3 enhancer element was knocked out in non-small lung cancer cell line A549 by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 （CRISPR/Cas9） technology.Flow cytometry,cell culture and PCR were used to identify knockout clones.Results Three homozygous 22q-Enh3 knockout clones were eventually obtained.Conclusion Our study provides a cell model for further evaluation of 22q-Enh3 function and what＆#39;s more,it accumulates valuable experience in the establishment of other knockout enhancer cell models with CRISPR/Cas9 technology.

关 键 词：CRISPR/Cas9 肺癌 基因敲除 增强子元件 

分 类 号：R392.11[医药卫生—免疫学]