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Construction of Transgenic Mice and Phenotypic Analysis of Tumor Suppressor PTEN |
LI Hong-chang, YUAN Lin, ZHANG Ling-qiang |
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China |
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Abstract 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 F0 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 from 3 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.
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Received: 24 March 2015
Published: 25 August 2015
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