摘要
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。
INK4a/ARF locus distinguishes itself by its unusual structure and function. It contains 2 overlapping genes with exons 1 α , 2 and 3 encoding p16 INK4a and exons 1 β ,2 and 3 encoding p19 ARF . Mice with their exons 2 and 3 of the INK4a/ARF knocked out are viable and fertile but develop spontaneous tumors at an early age and highly sensitive to carcinogenic treatment. However, mice with their exon 1 β knocked out, without interference the expression of p16 INK4a , show almost the same phenotype as those with their exons 2 and 3 knocked out. This raises a question of whether the mouse p16 INK4a plays a role in tumor suppression. To investigate this problem, a targeting vector pointing to p16 INK4a exon 1 α with 1.5kb Eco81Ⅰ/AccⅡ fragment as short arm and 5.9kb XbaⅠ/XhoⅠ fragment as long arm was built. After linearlization and purification, the targeting vector was introduced into ES cells through electroporation. Thirty seven G418 and gancyclovir resistant colonies were picked out and one of them was confirmed as positive by Southern hybridization.
基金
国家自然科学基金重点项目 ( 39830 36 0 )
上海联合利华科技和发展基金 ( 980 8)~~
