Construction and functional characterization of high mobility group box 2 gene knockout macro-phage Raw264.7 via clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 techno
Objective To construct a HMGB2 gene knockout mouse macrophage cell line(Raw264.7)with the CRISPR/Cas9 system,and verify whether HMGB2-/-Raw264.7 mediates the pro-duction of pro-inflammatory factors and bactericidal ability after Klebsiella pneumoniae(KP)infection.Methods Single guide RNA(sgRNA)was designed to target the mouse HMGB2 gene.The pX459 plas-mid inserted with the sgRNAs was transfected into Raw264.7 cells.Resistance screening and limiting dilu-tion methods were used to obtain HMGB2-/-Raw264.7 cell clones.Real-time polymerase chain reaction(RT-PCR)and Western blotting were used to identify the HMGB2 gene transcription and protein expression in the obtained cell clones.The ability of HMGB2-/-Raw264.7 cells to phagocytose and kill bacteria was measured by bacteria counting,and the secretion of pro-inflammatory factors by HMGB2-/-Raw264.7 cells was measured by enzyme-linked immunosorbent assay(ELISA).The t test was used for comparison between groups.Results Monoclonal cell lines were successfully screened using puromycin.HMGB2 pro-tein was not expressed at all in the HMGB2-/-Raw264.7 cell lines.HMGB2 knockout did not affect the macrophage phagocytosis of KP(2.37±0.31 vs.2.33±0.15,t=0.17,P＞0.05),but the survival rate of intracellular bacteria was significantly higher than that of wild type cell(WT)at 18 hpi.[(67.67±9.02)％vs.(32.33±6.11)％,t=5.62,P＜0.01];after KP infected HMGB2-/-Raw264.7 cells,the secretion of pro-inflammatory factors[tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6]was significantly lower than WT cells[(54.00±18.08)vs.(111.70±8.50)pg/ml,t=5.00,P＜0.01;(65.67±9.87)vs.(128.00±30.61)pg/ml,t=3.36,P＜0.01;(72.67±9.07)vs.(95.00±5.29)pg/ml,t=3.68,P＜0.05].Conclusion This study successfully constructed the HMGB2 gene knockout Raw264.7 cell line via CRISPR/Cas9 technology and verified the effect of HMGB2 on the induc-tion of inflammatory factors and resistance to KP infection.
CRISPR/Cas9 technologyHigh mobility group protein B2MacrophagesKlebsiella pneumoniae
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