建立血管内皮细胞敲除DEPTOR基因小鼠模型及鉴定  

Establishment and identification of a mouse model of vascular endothelial cell knockout DEPTOR gene

作　　者：丁燕 孟碧莹 向光大[1] Ding Yan;Meng Biying;Xiang Guangda(General Hospital of Middle Theater Command of Chinese PLA of Southern Medical University,Wuhan 430700,Hubei Province,China)

机构地区：[1]南方医科大学中国人民解放军中部战区总医院

出　　处：《中国组织工程研究》2019年第23期3698-3704,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81370896),项目负责人:向光大~~

摘　　要：背景:目前关于DEPTOR与血管类疾病的研究较少,在动物体内研究尚未发现,因此建立一种新的血管内皮特异性敲除DEPTOR的小鼠模型对于研究DEPTOR与血管类疾病的关系尤为重要。目的:建立血管内皮细胞敲除DEPTOR基因纯合子小鼠模型,并进行鉴定。方法:Cre小鼠,DEPTOR^flox/+雄鼠,均购于美国Jackson实验室;C57野生型小鼠购自华中科技大学。实验方案经中国人民解放军中部战区总医院动物实验伦理委员会批准(批准号:20120034)。选取5只DEPTOR^flox/+雄鼠与10只DEPTOR^flox/+雌鼠进行交配,得到基因型为EPTOR^flox/flox及DEPTOR^flox/+的F1子代小鼠35只,与8只血管内皮细胞特异性表达Tek^+重组酶的Cre小鼠进行交配,一共得到基因型为Tek-Cre^+×DEPTOR^flox/flox和DEPTOR^flox/flox子代小鼠共65只。采用PCR法进行DEPTOR^flox及Cre基因型鉴定,记录Tek-Cre^+×DEPTOR^flox/flox小鼠与DEPTOR^flox/flox小鼠2月龄的体长及体质量,采用Western blotting法检测2组小鼠肝脏组织DEPTOR蛋白相对表达量,免疫荧光检测两组小鼠血管内皮细胞DEPTOR的表达。结果与结论:①得到基因型为Tek-Cre^+×DEPTOR^flox/flox小鼠共25只、DEPTOR^flox/flox小鼠共40只,其体长分别为(18.61±1.14),(18.65±1.40)cm,体质量分别为(25.84±1.99),(25.06±2.15)g,二者比较差异均无显著性意义(均P>0.05);②Tek-Cre^+×DEPTOR^flox/flox小鼠与DEPTOR^flox/flox小鼠肝脏组织DEPTOR蛋白相对表达量分别为0.28±0.02,0.82±0.04,二者比较P<0.05;③血管内皮细胞DEPTOR阳染率分别为(73.67±2.87)%,(10.33±1.54)%,二者比较P<0.05;④结果证实成功敲除了Tek-Cre^+×DEPTOR^flox/flox小鼠的血管内皮细胞的DEPTOR基因;说明成功构建了血管内皮细胞敲除DEPTOR基因纯合子小鼠模型,并经基因型及蛋白组织水平鉴定。BACKGROUND:There are few studies on DEPTOR and vascular diseases,and no studies have been found in animals.Therefore,the establishment of a new mouse model of vascular endothelial specific knockout DEPTOR is important for studying the relationship between DEPTOR and vascular diseases.OBJECTIVE:To establish and identify a mouse model of vascular endothelial cell knockout DEPTOR gene.METHODS:Cre mice and DEPTOR^flox/+ mice were purchased from the Jackson Laboratory,and C57 mice were provided by Huazhong University of Science and Technology.The study was approved by the Animal Ethic Committee of General Hospital of Middle Thea ter Command of Chinese PLA,approval number:20120034.Five DEPTOR^flox/+male mice were selected to mate with 10 DEPTOR^flox/+ female mice,and 35 F1 progeny mice with genotype of EPTOR^flox/flox and DEPTOR^flox/+ were obtained and mated with 8 vascular endothelial cells specifically expressing Tek recombinase Cre mice.Finally 65 mice with genotypes of Tek-Cre^+× DEPTOR^flox/flox and DEPTOR^flox/flox progeny were obtained.The DEPTOR^flox and Cre genotypes were identified by PCR,and the body length and body mass of Tek-Cre^+× DEPTOR^flox/flox mice and DEPTOR^flox/flox mice at 2 months were recorded.The expression of DEPTOR protein in the mouse liver tissues was detected by western blot assay,and immunofluorescence was used to detect the expression of DEPTOR in the mouse vascular endothelial cells.RESULTS AND CONCLUSION:(1)There were 25 Tek-Cre^+× DEPTOR^flox/flox mice and 40 DEPTOR^flox/flox mice,with the body length of(18.61±1.14)and(18.65±1.40)cm,respectively,and body mass of(25.84±1.99)and(25.06±2.15)g,respectively(both P>0.05).(2)The relative expression level of DEPTOR protein in the Tek-Cre^+× DEPTOR^flox/flox mice and DEPTOR^flox/flox mice was 0.28±0.02 and 0.82±0.04,respectively(P<0.05).(3)The number of DEPTOR-positive cell in vascular endothelial cells was 73.67±2.87 and 10.33±1.54,respectively(P<0.05).(4)The results indicate that DEPTOR gene is successfully knocked out in Tek-C

关 键 词：血管内皮细胞 基因纯合子小鼠 Cre/loxp技术 DEPTOR基因 DEPTOR蛋白纯合子 基因敲除 

分 类 号：R446[医药卫生—诊断学] R318[医药卫生—临床医学]