摘要
目的:构建抑癌基因PTEN(phosphatase and tensin homolog deleted on chromosome ten)的转基因小鼠模型并对其表型进行初步分析。方法:细菌人工染色体(BAC)载体系统构建打靶载体创建PTEN转基因小鼠模型;利用对鼠尾DNA进行PCR检测的方法对出生的F0代小鼠进行基因型鉴定,将阳性F_0代小鼠与野生型小鼠交配繁殖筛选稳定遗传的转基因系。分离并培养小鼠胚胎成纤维细胞(mouse embryo fibroblast,MEF),利用Western blotting检测比较转基因阳性小鼠与同窝野生型小鼠MEF细胞中PTEN的蛋白表达水平,并通过克隆形成实验对比PTEN转基因小鼠与野生型小鼠MEF细胞的增殖能力;取成年小鼠主要组织器官提取蛋白,Western blotting检测PTEN转基因小鼠主要组织的PTEN蛋白表达情况;从小鼠出生后第三周开始统计、分析并制作小鼠的体重生长曲线;此外,还对比了PTEN转基因小鼠与野生型小鼠肺、肝、脾脏的细胞大小与腹腔内的脂肪含量。结论:PTEN转基因小鼠能够存活并稳定遗传;Western blotting结果表明,不论在胚胎期还是成年期,PTEN转基因小鼠体内的PTEN蛋白水平均高于同窝的野生型小鼠,转基因小鼠的PTEN表达水平接近野生型水平的3倍;对PTEN转基因小鼠的整体表型进行初步分析,发现Pten基因在体内过表达后,小鼠的体型显著变小,而细胞大小不变;腹腔内的脂肪含量显著减少。结论:成功构建了PTEN转基因小鼠模型,并获得了生理条件下PTEN过表达的原代细胞系,为研究抑癌基因PTEN的体内生理功能提供了重要的动物模型。
Objective： To establish transgenic mouse model of PTEN（ phosphatase and tensin homolog does on chromosome ten）,one of the most studied tumor suppressor genes in cancer field,and preliminarily analyze the phenotype of PTEN transgenic mouse model. Methods： The targeting vector of PTEN transgenic mouse model was designed through bacterial artificial chromosome（ BAC） carrier system,which can protect the inserted genes from position effect and ensure high expression of the inserted gene. PCR was used to identify the genotype of F0 mice and offspring. Positive transgenic mice in F_0 generation were crossed with wild type mice respectively to screen the stable transgenic mice line. At the same time. PTEN protein expression level was detected both in the embryonic and adult mice respectively,embryonic fibroblasts（ mouse embryo fibroblast,MEF） from PTEN transgenic mice and the wild type littermates were used to be detected and evaluated PTEN protein levels by Western blotting,and clone-formation assay was used to determine the MEF proliferation ability. Adult mice tissues of the high expression line were used to be a verification. Once confirmed PTEN was really over expression in the transgenic mice,weight of mice from the high expression transgenic line were recorded and analyzed from3 to 12 weeks. In addition,the cell size of liver,spleen and lung and abdominal fat were compared between PTEN transgenic and wile type MEFs. Results： PTEN transgenic mouse model was successfully constructed by taking advantage of BAC carrier system,genotyping the offspring of PTEN transgenic mice showed it can breed normally. Western blotting showed that PTEN expression levels were significantly higher in transgenic mice than in wild type ones,PTEN protein levels in embryonic fibroblasts cells（ MEF） from transgenic mice were as 3 folds as that from the wild type littermates,PTEN protein levels were also significantly higher in transgenic mice tissues than that in wild type ones. Moreover,the body size of PTEN transgenic mice is obviously smaller in the body size than that of wild type ones and intra-abdominal adipose content is significantly reduced. However,after comparing the cell size in liver,spleen and lung between transgenic mice and wile type mice,results showed that cells exhibit a same size. Conclusion： By using BAC strategy,PTEN transgenic mouse model was successfully established. Through isolating MEFs and evaluating PTEN level,cell lines stably overexpressing PTEN were smoothly obtained,which are very important for the study of tumor suppressor gene PTEN in physiological condition.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2015年第8期1-8,共8页
China Biotechnology
基金
国家自然科学基金重点项目(31330021)
北京市自然科学基金(5142020)资助项目
关键词
肿瘤
小鼠
转基因技术
PTEN
抑癌基因
Tumor Mouse Transgenic technology PTEN Tumor suppressor gene
