基于CRISPR-Cas9技术构建小鼠胚胎干细胞双荧光标记细胞系

Construction of double fluorescent labeled mouse embryonic stem cell line based on CRISPR-Cas9 tech⁃nique

目的构建p53-EGFP、Nanog-mcherry双荧光标记细胞系,对小鼠胚胎干细胞内p53、Nanog蛋白表达水平进行实时、定量追踪。在此基础上,初步探讨p53在小鼠胚胎干细胞中的作用。方法利用CRISPR-Cas9基因敲入系统分别将外源荧光蛋白表达基因插入到p53、Nanog基因组终止密码子后方,实现p53-EGFP、Nanog-mcherry融合表达。通过荧光强度分析,克隆形成能力检测等方法探索p53在小鼠胚胎干细胞中的作用。结果本研究成功构建了小鼠胚胎干细胞p53-EGFP、Nanog-mcherry双荧光标记细胞系,进一步发现p53蛋白表达在小鼠胚胎干细胞间具有异质性,且能调控Nanog蛋白表达的异质性。此外,p53蛋白表达量与小鼠胚胎干细胞克隆形成能力有关。结论基于CRISPR-Cas9基因敲入技术构建的双荧光标记细胞系中荧光强度的变化可以在活细胞状态下代表小鼠胚胎干细胞内相应蛋白表达水平,p53可通过其在小鼠胚胎干细胞群体间异质性表达参与小鼠胚胎干细胞转录调控网络中,并一定程度影响其自我更新能力。

Objectives To establish a double fluorescent labeled cell line with p53-EGFP,Nanog-mcherry fusion protein expression and to trace the real-time and quantitative expression levels of p53 and Nanog.Then we will investi⁃gate the functional roles of p53 in mouse embryonic stem cell.Methods The gene of exogenous fluorescent protein was inserted into the location following the stop codon of p53 or Nanog to generate the p53-EGFP and Nanog-mcherry fusion protein using CRISPR-Cas9 knock-in system.The roles of p53 in mouse embryonic stem cell were explored by fluorescence intensity analysis and clonogenic ability assay.Results In this study,we successfully established a new double fluorescent labeled cell line with p53-EGFP,Nanog-mcherry fusion protein expression in mouse embryonic stem cell,and revealed that p53 expression was heterogeneous among different cell populations,which could regulate the heterogeneity of Nanog expression.Furthermore,the expression level of p53 was related to the ability of mouse embryonic stem cell clone formation.Conclusions The change of fluorescence intensity in the double fluorescence labeled cell line based on CRISPR-Cas9 gene knockout technique can represent the corresponding protein expression level in mouse embryonic stem cells in the living cell state.p53 can participate in the transcriptional regulatory network of mouse embryonic stem cells through its heterogeneous expression in mouse embryonic stem cell populations,and affect its self-renewal ability to some extent.