Construction of a stable Atg5 gene knockdown cell line in RAW 264.7 cells by lentivirus infection

doi:10.3969/j.issn.2095-4344.2149

Abstract

Abstract: BACKGROUND: The autophagy of macrophage cells plays a key role in regulating the immune system and inflammatory response, while knockdown Atg5 gene can specifically inhibit the autophagy. There are some shortcomings of traditional siRNA transfection methods such as transience and incompleteness. It is of great value and significance to construct a stable Atg5 gene knockdown cell line in RAW 264.7 cells for studying the macrophage autophagy and immune inflammation-related diseases and their pathogenesis.
OBJECTIVE: To construct RAW 264.7 macrophage cell line stably knocking down the Atg5 gene by lentivirus infection, and to provide chassis cells for studying macrophage autophagy and immune inflammation-related diseases and their pathogenesis.
METHODS: Recombinant expression vector (HBLV-m-Atg5-shRNA-GFP-Puro) with green fluorescence signal, Puro resistance gene, and knockdown Atg5 gene was constructed and transfected into 293T cells to obtain lentivirus plasmid system. The green fluorescence signal of the acquired lentivirus infected RAW 264.7 cells was observed under an inverted fluorescence microscope. Purinomycin resistance screening and flow cytometry were used to obtain high-purity infected cells. The RAW 264.7 cell line stably knocking down the Atg5 gene was identified by real-time quantitative polymerase chain reaction and western blot assay.
RESULTS AND CONCLUSION: DNA sequencing results showed that Atg5 gene interfering sequences were correctly inserted into the expression vector, and the HBLV-m-Atg5-shRNA-GFP-Puro expression vector was successfully constructed. After infecting RAW 264.7 cell line with lentivirus plasmid, green fluorescence was observed under an inverted fluorescence microscope. Green fluorescent protein positive cell groups were observed by flow cytometry. The results of real-time quantitative polymerase chain reaction and western blot assay showed that the expression level of the Atg5 gene in RAW 264.7 cell line was significantly decreased (P < 0.05), indicating that the RAW 264.7 cell line with stable knockdown of Atg5 gene was successfully constructed.

Key words: lentivirus, gene, knockdown, autophagy, protein, cytology, cell group, macrophage

CLC Number:

Xian Guoyan, Chen Weishen, Zhang Ziji, Ye Yongyu, Pan Baiqi, Zheng Linli, Sheng Puyi. Construction of a stable Atg5 gene knockdown cell line in RAW 264.7 cells by lentivirus infection[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(1): 84-89.

Figures/Tables 6

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