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在活体水平上 ,小鼠p16 INK4a基因是否具有抑制肿瘤发生和发展的功能是一个悬而未决的问题。利用筛选基因组文库得到的小鼠p16 INK4a基因组DNA片段 ,构建了针对小鼠p16 INK4a 基因外显子 1α的基因打靶载体 ,其短臂为1.5kbEco81Ⅰ AccⅡ片段 ,长臂为 5 .9kbXbaⅠ XhoⅠ片段。打靶载体经线性化和纯化后通过电穿孔转导小鼠R1ES细胞 ,获得 37个G418和Gancyclovir双药抗性克隆。用Southern杂交法对双药抗性克隆进行鉴定 ,获得一个敲除了p16 INK4a基因外显子 1α的阳性ES细胞克隆。
At the living level, whether the mouse p16 INK4a gene has the function of inhibiting tumorigenesis and development is a pending issue. The gene targeting vector of mouse p16 INK4a gene exon 1α was constructed by screening the mouse p16 INK4a genomic DNA fragment obtained from the genomic library for screening. The short arm of the gene was 1.5 kb of EcoRI AccII and the long arm was a 5.9 kb XbaI XhoI fragment. Targeting vector transduced mouse R1 ES cells by electroporation after linearization and purification, obtaining 37 G418 and Gancyclovir two-drug resistant clones. Two-drug resistant clones were identified by Southern hybridization to obtain a positive ES cell clone knocked out of exon 1α of p16 INK4a gene.