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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 construct a transgenic mouse model of PTEN (phosphatase and tensin homolog deleted on chromosome ten) and analyze its phenotype. METHODS: The target vector was constructed by bacterial artificial chromosome (BAC) vector system to create PTEN transgenic mouse model. The genotypes of F0 generation mice were identified by PCR detection of mouse tail DNA. The positive F 0 generation mice and wild Mice were bred to screen stable transgenic lines. The mouse embryo fibroblast (MEF) was isolated and cultured. The protein expression of PTEN in transgenic mice and wild-type mice was detected by Western blotting. The expression of PTEN was compared with that of PTEN transgenic mice Mouse and wild-type mouse MEF cell proliferation; take the major mouse tissues of adult mice extract protein, Western blotting detection of PTEN transgenic mice major tissues of PTEN protein expression; from the third week after birth mice statistics, analysis and analysis The body weight growth curve of mice was also made. In addition, the lung, liver and spleen cell size and the intra-abdominal fat content in PTEN transgenic mice and wild-type mice were also compared. CONCLUSION: PTEN transgenic mice survived and remained stable. The results of Western blotting showed that the PTEN protein levels in PTEN transgenic mice were higher than those in wild-type mice and PTEN transgenic mice both in embryonic and adulthood The expression level of PTEN transgenic mice was nearly 3 times that of the wild type. The overall phenotype of PTEN transgenic mice was analyzed preliminarily. It was found that the body size of mice was significantly reduced and the cell size was unchanged after the Pten gene was overexpressed in vivo. Significantly reduced content. CONCLUSION: The transgenic mouse model of PTEN was successfully constructed and the primary cell line PTEN overexpressed under physiological conditions was obtained. It provides an important animal model for studying the in vivo physiological function of tumor suppressor gene PTEN.