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

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×FLAG tags into the front of SND1 gene in HeLa cells, so that the endogenous expression of SND1 protein in cells was equipped with 3×FLAG tags, and the localization of SND1 with stress granules and processing bodies was observed. A sgRNA near the start codon ATG of the SND1 gene was designed, and a recombinant eukaryotic expression plasmid was constructed using px459 as an expression vector. A sequence containing 3×FLAG 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 3×FLAG-SND1 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 eIF2α 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 DCP1α. © 2019 The authors.