Construction and characterization of mice with conditional knockout of Stat3 gene in microglia

Abstract

Objective·To construct mice with conditional knockout of Stat3 gene in microglia based on the Cre-Loxp system and validate their knockout efficiency.Methods·Cx3cr1creERT2 and Stat3fl/fl genotypic mice were bred for conditional knockout mice (CKO) and Wild Type mice (WT). The mouse genotypes were determined by extracting DNA from mouse tissues through Polymerase Chain Reaction (PCR) combined with the amplification results of cre and flox primers. Stat3 knockdown was induced by intraperitoneal injection of tamoxifen in the CKO and WT mice at 6 weeks of age. The CKO mice (n=4) and WT mice (n=4) were randomly selected for the detection. After two weeks of observation, microglia cells were sorted out by Magnetic Activated Cell Sorting (MACS). Real-time PCR (RT-PCR) was used to detect gene knockout efficiency at the gene level. The expression of STAT3 in microglia was observed by brain immunofluorescence staining. The expression rate of STAT3 in microglia was detected by flow cytometry. The expression rate of STAT3 in macrophages of the spleen was detected by flow cytometry. The condition of neuronal cells was examined by Nissl staining. The condition of the microglia in the cortex and hippocampus was observed by brain immunofluorescence staining. The phenotype of the microglia was detected by flow cytometry.Results·The CKO mice and WT mice were successfully bred. MACS boosted the proportion of microglia in brain cells from 10% to 85%. RT-PCR results showed that mRNA levels of Stat3 were down-regulated in microglia of CKO mice, compared with the WT mice (P=0.001). The relative mRNA expression of Stat3 in microglia of the CKO mice was 0.331 7±0.041 4. Immunofluorescence staining of brain tissues showed that the fluorescence intensity of STAT3 in microglia of the CKO mice was weaker than that of the WT mice. Flow cytometry of brain tissues showed that the STAT3-positive cells in microglia of the WT mice was (85.30±5.69)% and the CKO mice was (39.70±3.88)%. STAT3 expression was decreased in microglia of the CKO mice (P=0.001). Flow cytometry of spleen tissues showed that there was no statistical difference in the percentage of STAT3-positive cells in splenic macrophages between the CKO and WT mice (P>0.05). Nissl staining showed that there were no significant differences between the neuronal cells of the CKO mice and WT mice. Immunofluorescencestaining of brain tissues showed that there was no significant difference in the shape of microglia between the CKO mice and WT mice. Flow cytometry showed that the phenotype of microglia in the CKO mice was not remarkably different from that of the WT mice.Conclusion·We successfully construct the STAT3 gene conditional knockout mice from microglia, which provides the foundation for subsequent related studies.

Keywords

• microglia
• signal transducer and activator of transcription 3 (stat3)
• tamoxifen
• mice
• cre recombinase