DDR2血管内皮细胞条件性基因敲除小鼠的复制、鉴定及表型分析*

Generation, genotyping and phenotype analysis of DDR2 endothelial cell conditional knockout mice*

摘要:

目的 应用Cre/LoxP系统复制盘状结构域受体2（DDR2）在血管内皮细胞可诱导性条件性基因敲除小鼠模型DDR2i?EC（DDR2flox/flox，Cdh5-Cre/ERT2），并分析其在肝脏中的表型。方法 复制DDR2打靶载体，通过电转入小鼠胚胎干细胞（ES细胞）打靶，通过聚合酶链反应（PCR）+脱氧核糖核酸（DNA）印迹法鉴定筛选阳性ES细胞，将阳性的ES细胞注射到C57BL/6N小鼠囊胚，移植入受体小鼠子宫以获得嵌合体小鼠。将得到的嵌合体小鼠与C57BL/6N小鼠回交得到F0杂合子，F0代杂合小鼠自交筛选获得F1代DDR2flox/flox小鼠，该小鼠与Cdh5-Cre/ERT2小鼠杂交，通过子代自交获得DDR2在血管内皮细胞可诱导性条件性基因敲除（DDR2flox/flox， Cdh5-Cre/ERT2）小鼠。他莫昔芬诱导血管内皮细胞上DDR2基因敲除，对小鼠进行表型分析。结果 获得DDR2血管内皮细胞可诱导性条件性基因敲除小鼠DDR2i?EC（DDR2flox/flox，Cdh5-Cre/ERT2），DDR2i?EC小鼠出生时符合孟德尔遗传定律，发育良好，他莫昔芬诱导敲除小鼠血管内皮细胞DDR2基因表达后，小鼠出现自发性肝纤维化及黄体血管新生障碍等表型。结论 成功复制DDR2血管内皮细胞可诱导性条件性基因敲除小鼠模型。血管内皮细胞DDR2缺失能导致自发性肝纤维化。

Abstract:

Objective To create a tamoxifen induced discoidin domain receptor 2 (DDR2) endothelial conditional knockout mouse line DDR2i?EC (DDR2flox/flox; Cdh5-Cre/ERT2) by Cre-LoxP system and to analyzed the phenotype in liver. Methods DDR2 conditional knockout targeting vector was constructed and then transfected into mouse embryonic stem (MES) by electroporation. The successfully transferred MES cells were screened by PCR and Southern-blotting to identify positive cells. The positive targeted MES cells were microinjected into the blastula of C57 BL/6N mice and chimeric mice were generated after transplanting the blastula into the host mice. After crossing breeding between the chimeric and C5BL/6N mice to get F0 heterozygote, which inbred and were screened, DDR2flox/flox mice were generated. DDR2 Endothelial cell conditional knockout mice (DDR2flox/flox; Cdh5-Cre/ERT2) could be further gained by crossing breeding between Cdh5-Cre/ERT2 transgenic and DDR2flox/flox mice. The DDR2 gene deletion on vascular endothelial cells was achieved by tamoxifen induction, and the phenotypes of mice were analyzed. Results We had acquired DDR2i?EC (DDR2flox/flox; Cdh5-Cre/ERT2) mice successfully. The deletion of DDR2 gene expression on vascular endothelial cells was successfully induced by tamoxifen. DDR2i?EC mice were comparable with Mendelian’s law at birth and can grow up with normal development. DDR2i?EC mice showed spontaneous liver fibrosis and luteal angiogenesis disorder compared to WT mice. Conclusions We have successfully generated a DDR2 endothelial specific knockout mouse line and find endothelial DDR2 deficiency in mice induced spontaneous liver fibrosis, which lay a foundation for later learning on the molecular function of DDR2 in vascular endothelial cell.