小鼠LSK及DN原代细胞中基因编辑方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2017-0903

生物技术通报 ›› 2018, Vol. 34 ›› Issue (5): 64-70.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0903

小鼠LSK及DN原代细胞中基因编辑方法的建立

王跃强^1,2,AvinashBhandoola¹

1. Laboratory of Genome Integrity,Center for Cancer Research,National Cancer Institute,Bethesda,MD 20892-4254,USA;
2. 深圳国家基因库合成与编辑平台,深圳 518083

收稿日期:2017-10-25 出版日期:2018-05-26 发布日期:2018-06-07
作者简介:王跃强,博士,研究方向：遗传学;E-mail：wangyueqiang@cngb.org
基金资助:
美国国立卫生研究院（NIH）（AI059621,AI098428）

Gene Editing in Mouse Lin^-Sca1⁺Kit⁺ and Double Negative（DN）Primary Cell

WANG Yue-qiang^{1, 2}, Avinash Bhandoola¹

1. Laboratory of Genome Integrity,Center for Cancer Research,National Cancer Institute,Bethesda,MD 20892-4254,USA;
2. Genome Synthesis and Editing Platform,China National GeneBank,Shenzhen 518083

Received:2017-10-25 Published:2018-05-26 Online:2018-06-07

摘要/Abstract

摘要： 小鼠（Mus musculus）免疫系统包含种类众多、功能各异的细胞群体。为了研究小鼠免疫系统的发育机制,需要建立高效的遗传操作技术方法。首先选择Cas9敲入小鼠的DN细胞（CD4^-CD8^-double negative thymocytes）为研究对象,针对GFP和CD4表面抗原基因设计构建了sgRNA（single guide RNA）,使用小鼠逆转录病毒（Murine stem cell virus,MSCV）为载体实施了基因编辑。通过流式细胞仪分析,检测到GFP和CD4被成功敲除。在此基础上,选取Cas9敲入小鼠的LSK细胞（Lin^-Sca1⁺Kit⁺）为研究对象,使用同样的方法针对Tcf7基因设计构建了sgRNA并实施基因编辑。基因编辑后,LSK细胞不能正常分化为下游T细胞,但其分化为髓系细胞能力不受影响。这些结果表明,针对小鼠LSK和DN原代细胞实施的基因编辑取得成功。

关键词: 小鼠, 免疫, 干细胞, 基因编辑, CRISPR/Cas9

Abstract: The immune system of mice（Mus musculus）contains a large number of cell groups with different functions. In order to study the development mechanism of mouse immune system,it is necessary to establish efficient genetic manipulation techniques. Firstly CD4^-CD8^- double negative thymocytes（isolated from Cas9 knock-in mice）was chosen for this study,then sgRNAs（single guide RNA）were designed to target GFP and mouse CD4 cell surface antigen genes,and murine stem cell virus was used as a vector to deliver sgRNA into the cell,and by which gene editing was realized. The successful knockout of GFP and CD4 were detected by flow cytometry analysis. Based on these results,using Lin^-Sca1⁺Kit⁺ cell（isolated from Cas9 knock-in mice）as study materials,sgRNAs were designed and constructed with the same method for gene editing. After gene editing,the LSK cells did not differentiate into downstream T cells,but their myeloid differentiation potential was not affected. All these results suggest that we succeed in establishing mouse DN and LSK primary cell gene-editing platform.

Key words: mouse, immunology, stem cell, gene editing, CRISPR/Cas9

王跃强,AvinashBhandoola. 小鼠LSK及DN原代细胞中基因编辑方法的建立[J]. 生物技术通报, 2018, 34(5): 64-70.

WANG Yue-qiang, Avinash Bhandoola. Gene Editing in Mouse Lin^-Sca1⁺Kit⁺ and Double Negative（DN）Primary Cell[J]. Biotechnology Bulletin, 2018, 34(5): 64-70.

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小鼠LSK及DN原代细胞中基因编辑方法的建立

Gene Editing in Mouse Lin^-Sca1⁺Kit⁺ and Double Negative（DN）Primary Cell

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小鼠LSK及DN原代细胞中基因编辑方法的建立

Gene Editing in Mouse Lin-Sca1+Kit+ and Double Negative（DN）Primary Cell

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Gene Editing in Mouse Lin^-Sca1⁺Kit⁺ and Double Negative（DN）Primary Cell