@misc{oai:ir.soken.ac.jp:00001082,
 author = {渡部, 聡 and ワタナベ, サトシ and WATANABE, Satoshi},
 month = {2016-02-17, 2016-02-17},
 note = {I developed a novel method for production of transgenic mice for the phenotypic rescue experiment of gene knockout mice with ES cells. For this experiment, I showed that puromycin, a protein synthesis inhibitor, could be used for the selection of recombinant ES cells from heterozygously gene disrupted G418 resistant ES cells. ES cells were killed by culturing with purornycin at a concentration O. 1 mg/ml for 2-days. Puromycin could function independently from G418, because G418 resistant ES cells were also killed at the same condition. ES cells could acquire puromycin resistance by introducing the pac gene, and the recombinant ES cells could survive against the puromycin selection. G418-puromycin double drug resistant ES cells could generate chimeric mice at high rate, and maintained high germline differentiating potency. The pac gene was transmitted to offsprings via chimeric mice, and the gene function was maintained among them. <br />  For the rescue experiment by the gene-trap method, I used Fyn knockout mice as a  model. GT-2 gene-trap vector was introduced into fyn heterozygously disrupted ES cells. The vector contained the lacZ gene and hurman fyn cDNA as reporter genes and the pac gene as a selection marker. Among puromycin resistant ES cells, lacZ positive clones were obtained. In these clones, Fyn was also expressed, and the insertions of the GT-2 vector were confirmed by southern blotting analysis. These results suggested that the endogenous promoter could direct the expression of introduced gene. Chimeric mice were produced with the lacZ positive clones by the microinjection, and germline chimeras were obtained from three clones. The mice with the subjected genotype [fyn(-/-),gt/+] were obtained among F1 offsprings between these chimeras and Fyn deficient mice., application/pdf, 総研大甲第160号},
 title = {マウス胚幹細胞を用いた新たなトランスジェニックマウス作製技術の開発},
 year = {}
}