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

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。

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 designedto 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,andby 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 methodfor 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.