小鼠adiponectin的甲基化敏感性检测及其启动子克隆

The methylation sensitive detection of adiponectin and it’s promoter clonning

本试验通过对脂肪组织特异表达的脂肪因子adiponectin调控区域4个CpG位点在心、肝、脾、肺、肾、骨骼肌、腹脂、内脏脂肪中的甲基化程度以及adiponectin mRNA表达水平的检测,表明adiponectin在mRNA水平的表达与其调控位点的甲基化程度显著相关;同时采用PCR技术克隆了小鼠脂肪组织特异表达的脂联素的启动子,通过DNA重组技术将该基因的启动子重组于pEGFP-N1真核表达载体上,构建pEGFP-N1-P(adi)重组质粒,通过分子克隆测序方法对重组质粒进行鉴定,并转染小鼠前脂肪细胞,通过荧光素酶活性检测表明P(adi)具备驱动报告基因表达的活性,为今后制备转基因动物的研究奠定基础。

Through the detection of adiponectin regulatory regions four CpGss in heart,liver,spleen,lung,kidney,skeletal muscle and abdominal fat,visceral fat in the degree of methylation and mRNA expressing level,chich indicate a significa correlation between the methylation of CpG sites and mRNA expressing level.Then,cloned mice adipose tissue specific expression of adiponectin promoter by PCR technology,by recombinant DNA technology will be the gene promoter restructuring in pEGFP-N1 eukaryotic expression vector,which pEGFP-N1-P（adi） recombinant plasmid,through the molecular cloning sequencing methods recombinant plasmid were identified,and transfection adipose-derived stem cells,through the luciferase activity test show that P（adi） have driven report gene expression activity,for the preparation of transgenic animal lays the foundation.