应用CRISPR/Cas9技术构建内源表达双标签NPM1融合蛋白的细胞系

Construction of cell lines expressing NPM1 fusion protein with two tags using CRISPR/Cas9 system

目的 旨在创建一个内源表达NPM-Stag-Flag融合蛋白的细胞模型,为精确分离和鉴定未知NPM1互作蛋白奠定基础。方法 结合CRISPR/Cas9基因编辑技术并利用同源重组机制在内源NPM1基因位点敲入编码Stag-Flag的序列,通过药物筛选分离和鉴定单克隆细胞。结果 成功建立了两种纯合型敲入Stag-Flag的人T淋巴瘤JurkatE6-1细胞系。在C-端插入Stag-Flag标签的NPM1表达效率明显优于N-端插入型。利用双标签的两步亲和纯化法可有效富集到NPM1蛋白复合物。结论 内源表达NPM1-Stag-Flag的JurkatE6-1细胞系是今后寻找未知NPM1互作分子和开展功能研究的有力工具。

Objective To create a cell model of endogenous expression of NPM-Stag-Flag fusion protein to lay a foundation for accurate isolation and identification of novel NPM1 interacting proteins.Methods Combined with CRISPR/Cas9 gene editing technology and homologous recombination mechanism,the sequence encoding Stag-Falg was knocked into the endogenous NPM1 gene locus,and monoclonal cells were isolated and identified through drug screening.Results Two homozygous human T lymphoma JurkateE6-1 cell lines with stag-flag knockin were successfully established.NPM1 expression efficiency of Stag-flag tag inserted in C-terminal is significantly better than N-terminal insertion type.NPM1 protein complex can be enriched effectively by two-step affinity purification with dual label.Conclusion JurkateE6-1 cell line with endogenous expression of NPM1-Stag-Flag is a powerful tool to search for novel NPM1 interacting molecules and to carry out functional research in the future.