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  • 袁媛,张冠宇,孙平新,章越凡,李铁军,李文林.利用CRISPR/Cas9系统建立叉头框G1基因敲除的人胚胎干细胞系[J].第二军医大学学报,2020,41(5):513-519    [点击复制]
  • YUAN Yuan,ZHANG Guan-yu,SUN Ping-xin,ZHANG Yue-fan,LI Tie-jun,LI Wen-lin.Establishment of human embryonic stem cell line with forkhead box G1 gene knockout by CRISPR/Cas9[J].Acad J Sec Mil Med Univ,2020,41(5):513-519   [点击复制]
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利用CRISPR/Cas9系统建立叉头框G1基因敲除的人胚胎干细胞系
袁媛1,2,张冠宇3,孙平新3,章越凡2,李铁军1,2,李文林3*
0
(1. 安徽中医药大学研究生院药学系, 合肥 230012;
2. 海军军医大学(第二军医大学)药学院药理学教研室, 上海 200433;
3. 海军军医大学(第二军医大学)基础医学院细胞生物学教研室, 上海 200433
*通信作者)
摘要:
目的 采用成簇的规律间隔的短回文重复序列及其相关蛋白9(CRISPR/Cas9)基因编辑技术构建叉头框G1(FOXG1)基因敲除的人胚胎干细胞系,研究FOXG1基因在人胚胎干细胞早期神经诱导过程中的作用。方法 利用CRISPR/Cas9基因编辑技术,通过转染2个向导RNA(gRNA)诱导人胚胎干细胞FOXG1基因的大片段敲除,经单克隆筛选、测序分析和蛋白质印迹分析验证获得FOXG1基因敲除的人胚胎干细胞;通过细胞免疫荧光染色、qRT-PCR检测FOXG1基因敲除前后细胞在早期神经诱导过程中关键标志物配对框基因6(PAX6)、性别决定区Y框蛋白2(SOX2)和正小齿同源物2(OTX2)的表达。结果 利用CRISPR/Cas9基因编辑技术成功获得FOXG1基因大片段缺失的人胚胎干细胞,细胞免疫荧光染色、qRT-PCR结果均显示人胚胎干细胞早期神经诱导过程中的关键标志物PAX6、SOX2和OTX2的表达并未受FOXG1缺失的影响。结论 通过2个gRNA共转染可以快捷地诱导人胚胎干细胞FOXG1基因的大片段敲除。FOXG1基因缺失并不影响人胚胎干细胞的早期神经诱导。
关键词:  CRISPR/Cas9基因编辑  叉头框蛋白G1  基因敲除  人胚胎干细胞
DOI:10.16781/j.0258-879x.2020.05.0513
投稿时间:2020-02-10修订日期:2020-04-13
基金项目:国家自然科学基金面上项目(81571094),上海市科学技术委员会优秀学术带头人计划(17XD1404800).
Establishment of human embryonic stem cell line with forkhead box G1 gene knockout by CRISPR/Cas9
YUAN Yuan1,2,ZHANG Guan-yu3,SUN Ping-xin3,ZHANG Yue-fan2,LI Tie-jun1,2,LI Wen-lin3*
(1. Department of Pharmacy, Graduate School, Anhui University of Chinese Medicine, Hefei 230012, Anhui, China;
2. Department of Pharmacology, School of Pharmacy, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
3. Department of Cell Biology, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To construct human embryonic stem cell (hESC) line with forkhead box G1 (FOXG1) gene knockout by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology, and to investigate the role of FOXG1 gene in the early neural induction of hESCs. Methods Two guide RNAs (gRNAs) were transfected to induce FOXG1 gene large fragment knockout in hESCs by CRISPR/Cas9 gene editing technology. FOXG1 gene knockout hESCs were confirmed by monoclonal screening, sequencing and Western blotting analysis. The expression of the key markers including paired box 6 (PAX6), sex-determining region Y-box 2 (SOX2) and orthodenticle homeobox 2 (OTX2) was detected by immunofluorescence staining and qRT-PCR in the early process of neural induction before and after FOXG1 gene knockout. Results hESCs with FOXG1 gene large fragment knockout were successfully obtained by CRISPR/Cas9 gene editing technology. The results of immunofluorescence staining and qRT-PCR suggested that FOXG1 deletion did not significantly influence the expression of PAX6, SOX2 and OTX2 during neural induction. Conclusion FOXG1 gene large fragment knockout in hESCs can be quickly induced by a pair of gRNAs co-transfection. FOXG1 deletion has no significant impacts on neural induction of hESCs.
Key words:  CRISPR/Cas9 gene editing  forkhead box protein G1  gene knockout  human embryonic stem cells