基因前定点敲入FLAG标签表达的FLAG-SND1蛋白参与胞内应激颗粒的形成

The FLAG-SND1 Protein Whose FLAG Tag was Knocked in the Front of the Gene was Involved in the Formation of Intracellular Stress Granules

CRISPR/Cas9(clustered regularly interspaced short palindromic repeats-Cas9 nuclease)基因编辑技术是近年来新兴的一种可以实现基因特异性敲除和敲入的技术。本文利用CRISPR/Cas9基因编辑系统,将3×FLAG标签定点敲入HeLa细胞SND1基因前方,使细胞内源性表达的SND1蛋白带有3×FLAG标签,并观察SND1与应激颗粒及加工体的定位情况。设计针对SND1基因起始密码子ATG附近的sgRNA,以px459为表达载体,构建出重组真核表达质粒。设计含有3×FLAG及待插入位置上下游150 bp同源臂的序列,经公司合成获得重组质粒。将2个质粒共同转染HeLa细胞,使用嘌呤霉素筛选阳性细胞,挑取单克隆后培养。Western印迹表明,细胞表达3×FLAGSND1融合蛋白质。提取细胞基因组DNA进行测序。测序无误获得稳定株后,用流式细胞术检测细胞周期和凋亡,发现与WT细胞相比无显著性差异。同时,使用05 mmol/L亚砷酸钠处理,细胞发生氧化应激,eIF2α蛋白磷酸化增加,胞浆中出现应激颗粒,SND1与应激颗粒标志蛋白TIAR存在共定位现象,但不存在与加工体蛋白DCP1α的共定位。

CRISPR/Cas9(clustered regular interspaced short palindromic repeats-Cas9 nuclease)gene editing technology is an emerging technology for gene-specific knock-out and knock-in in recent years.In this experiment,the CRISPR/Cas9 gene editing system was used to insert 3 xFLAG tags into the front of SND\gene in HeLa cells,so that the endogenous expression of SND1 protein in cells was equipped with 3xFLAG tags,and the localization of SND1 with stress granules and processing bodies was observed.A sgRNA near the start codon ATG of the 5A^D1 gene was designed,and a recombinant eukaryotic expression plasmid was constructed using px459 as an expression vector.A sequence containing 3xFLAG and a 150 bp homologous arm upstream and downstream of the position to be inserted was designed,and the recombinant plasmid was synthesized by the company.Two plasmids were co-transfected into HeLa cells,and positive cells were screened by puromycin.Western blotting indicated that the cells expressed the 3xFLAG-SNDl fusion protein.Genomic DNA was extracted and sequenced.Cell cycle and apoptosis were detected by flow cytometry after sequencing and the stable strain was obtained without error.It was found that there was no significant difference compared with WT cells.At the same time,the treatment with 0.5 mmol/L sodium arsenite resulted in oxidative stress in cells,increased phosphorylation of eIF2a protein,and the presence of stress particles in the cytoplasm.There was co-localization between SND1 and stress particle marker protein TIAR,but no co-localization with processed body protein D C P la.