Stable knockout of Mcart-1 down-regulates the proliferation and oxidative phosphorylation of RAW264.7 macrophages

• Published in: Ji chu yi xue yu lin chuang = Jichu yixue yu linchuang = Basic medical sciences and clinics Vol. 43; no. 4; pp. 568 - 575
• Main Author: ZHAO Yongjing, QIU Jiaxing, ZHANG Diya, GUO Hongjiang, WANG Yucheng, JU Rui, GUO Lei
• Format: Journal Article
• Language: Chinese
• Published: Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College 01-04-2023
• Subjects: crispr/cas9|macrophage|mcart-1|metabolic phenotype
• ISSN: 1001-6325
• DOI: 10.16352/j.issn.1001-6325.2023.04.0568

Objective To construct a RAW264.7 macrophage cell strain with stable knockout of Mcart-1 by using CRISPR/Cas9 gene editing technique, and to detect its biological function. Methods Cas9 and sgRNA lentivirus were used to infect RAW264.7 macrophages in two steps. Positive cells were screened with puromycin and hygromycin, and single cells were plated by flow cytometry to obtain monoclonal cells; Expression of Cas9 and Mcart-1 was detected by qPCR and Western blot, and the mutation site was confirmed by sequence analysis. Cells number counting and carboxyfluorescein diacetate succinimdyl ester (CFSE) staining were used to detect cell proliferation; ATP detection kit was used to measure total ATP content in cells; Seahorse bioenergy analyzer was used to detect cell oxygen consumption rate(OCR) and glycolysis rate. Results Successfully constructed RAW264.7 cell strain with Mcart-1 stably knocked out, denoted as Mcart-1-/--RAW264.7; A frameshift mutation occurred in Mcart-1 gene in this cell strain; Compared with t