利用同源重组对酵母人工染色体左右臂进行多基因修饰

构建携带哺乳动物细胞筛选基因和酵母人工染色体(YAC)同源序列的载体,利用酵母中能够发生高频率同源重组的特点对YAC分别进行左、右臂修饰,依次将NEO、EGFP及PURO基因定点整合到YAC左右臂上。用营养缺陷筛选的方法排除酵母发生突变或随机整合等情况后,用PCR及Southern杂交方法证实各筛选基因定点整合于YAC两臂上,从而获得携带3个哺乳动物细胞筛选基因的YAC克隆。并且由此建立了通过同源重组将哺乳动物标记基因定点引入YAC左右臂的多基因修饰平台。

Modification of YAC Both Arms with Three Genes by Homologous Recombination

Yeast artificial chromosome (YAC) was widely used in genomic and chromosome relative researches due to its extraordinary capacity of loading large DNA segments (megabase pairs, Mbp). In this experiment, different vectors bearing YAC homologous sequences and mammalian cell selective genes, such as NEO, EGFP, PURO, were transferred into yeast. By means of homologous recombination, the YACs, which were modified by the genes mentioned above on certain fragments of both arms, were selected and pooled on nutrition-deficient medium, excluded mutation and random integration. Then, the integration of NEO, EGFP and PURO into YAC DNA were identified using PCR and Southern blotting methods. The results indicated that such integration was successfully carried out and the YAC bearing mammalian selective genes was obtained. At the same time, the method, which mammalian selective genes integrated on YAC arms using homologous recombination, was constructed.