应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成

doi:10.11843/j.issn.0366-6964.2020.09.025

Abstract

Abstract: When mammalian cells are stimulated by stress, multiple dense granular structures form in cells. These structures are called stress granules (SGs). G3BP1 is an important component and marker protein of stress granules. To dynamically monitor the formation of SG, the HeLa cell line stably expressing GFP-G3BP1 was constructed. The G3BP1 gene was amplified and cloned into a lentiviral vector to obtain a recombinant plasmid Lenti-GFP-G3BP1. The three-plasmid lentiviral packaging system was used to package the lentiviral particles expressing GFP-G3BP1, which were used to infect HeLa cells. The positive cells were initially selected by puromycin, and further selected by limited dilution to get the monoclonal cells stably expressing GFP-G3BP1. The function and expression of GFP-G3BP1 in the cell line were detected by Western blot and immunofluorescence assays. The obvious cytoplasm-specific fluorescence of G3BP1 was observed. Western blot results show a GFP-G3BP1 specific band, indicating that the HeLa cell line stably expressing GFP-G3BP1 was constructed successfully. Finally, the formation of SG in response to ARS/heatshock/Newcastle disease virus treatment were dynamically monitored. The results showed the gradual accumulation of SGs in response to these treatments. The specific cell line and method we established have laid the foundation for subsequent research on SG and Newcastle disease virus.

Key words: G3BP1, stress granule, Newcastle disease virus, virus infection

CLC Number:

S855.3

SHAO Qi, QU Yang, ZHU Zichen, MENG Chunchun, QIU Xusheng, LIAO Ying, TAN Lei, SONG Cuiping, LIU Weiwei, SUN Yingjie, DING Chan. Monitoring of Stress Granule Formation under Stress by G3BP1 Stable Expressing Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2275-2283.

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Monitoring of Stress Granule Formation under Stress by G3BP1 Stable Expressing Cell Line

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