Construction and Identification of a Macrophage-specific Colgalt1 Gene Knockout Mouse Model
10.13865/j.cnki.cjbmb.2025.05.1020
- VernacularTitle:巨噬细胞特异性胶原β(1-O)半乳糖基转移酶1基因敲除小鼠模型的构建与鉴定
- Author:
Pei-Pei QIU
1
;
Xiao-Jiao SUN
;
WANG-LEI
;
Zhi-Qi WANG
;
Chu-Xiao YI
;
Zhen-Ming LIU
;
Ji-Guo ZHANG
Author Information
1. 山东第一医科大学(山东省医学科学院)药学院(药物研究所),济南 250117
- Publication Type:Journal Article
- Keywords:
collagen β(1-O)galactosyltransferase 1(colgalt1);
conditional gene knockout;
macro-phage;
cre-LoxP recombinase system;
animal model
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(8):1214-1222
- CountryChina
- Language:Chinese
-
Abstract:
Aberrant expression of Colgalt1 is closely associated with tumorigenesis and tumor progres-sion;however,the mechanism by which it regulates macrophages to influence tumor development remains poorly understood.This study aimed to establish a macrophage-specific Colgalt1 gene knockout mouse model to delve into the mechanisms through which Colgalt1 modulates macrophage function and subse-quently affects the occurrence and progression of tumor-related diseases.Initially,Colgalt1flox+mice were generated using gene editing techniques,followed by crossing with Lyz2-Cre+mice,which exhibit tissue-specific expression in the myeloid lineage(including monocytes and mature macrophages).Through this strategy,mice with the genotype Colgalt1-/-Lyz2-Cre+were successfully obtained,achieving conditional knockout of the Colgalt1 gene in macrophages.Colgalt1flox/flox Lyz2-Cre-mice were used as control.PCR and agarose gel electrophoresis were employed to identify the Flox and Cre genotypes of the knockout mice.RT-qPCR and Western Blot techniques were utilized to detect the expression levels of Colgalt1 in BMDMs from knockout mice at both the mRNA and protein levels,respectively.Western Blot results re-vealed a significant downregulation of Colgaltl expression in BMDMs from knockout mice compared to controls(P＜0.01).RT-qPCR results demonstrated a significant reduction in Colgalt1 mRNA levels in BMDMs from knockout mice compared to contro1s(P＜0.001),while no significant differences in Col-galt1 mRNA expression were observed in liver,lung,or spleen tissues between the two groups.Addition-ally,immunohistochemistry was employed to detect Colgalt1 expression in liver-specific macrophages,re-vealing an absence of Colgalt l-positive staining in liver macrophages from knockout mice.HE staining was used to observe cellular morphology in liver tissues from both groups of mice,showing no significant differences in cellular morphology or obvious pathological changes in tissues and organs.Moreover,the o-verall survival of the mice was not affected.Finally,RT-qPCR was used to assess the expression of mac-rophage-related inflammatory factors in BMDMs from both groups of mice.The results indicated that com-pared to controls,knockout mice exhibited downregulated expression of TNF-α(P＜0.05)and signifi-cantly upregulated expression of IL-10(P＜0.01),Arginase1(P＜0.001),and CD206(P＜0.001)in BMDMs,suggesting an anti-inflammatory trend and M2 polarization of macrophages following Colgalt 1 knockout.In summary,this study successfully established a macrophage-specific Colgalt1 gene knockout mouse model,providing a more reliable experimental animal model for in-depth exploration of the specific roles of Colgalt1 in macrophage functional regulation and the pathogenesis of tumor-related diseases.This model holds promise for identifying novel therapeutic targets and strategies for tumors and other diseases.
