Red/ET同源重组介导细菌人工染色体的快速修饰

Rapid Modification of BACs With The Optimized Red/ET Recombination System

随着基因组测序工程的实施与完成,如何对包含完整基因信息的特定细菌人工染色体(BAC)进行有目的修饰,已成为功能基因组学研究的一个重要环节.应用新近优化的Red/ET同源重组技术对目标BAC进行修饰,以pSC101-BAD-gbaA为依托质粒,采用rpsL鄄neo为正/反向筛选系统,可以快速、高效地对BAC进行剪切、插入、替换等操作,其中能够进行抗性筛选的一步BAC修饰只需一周时间,以插入非抗性标记基因Cre为代表的两步BAC修饰在两周内即可完成.通过阿拉伯多糖诱导调控和简单地变化培养温度,能使pSC101-BAD-gbaA依托质粒在发挥完Red/ET同源重组作用后自然消失,最终获得完整而纯净的修饰后BAC,为加快功能基因组学研究提供了一个可靠的实验平台.

With the completion of genome-sequencing projects, intentional modification of definite bacterial artificial chromosomes (BACs), which carry entire components of most eukaryotic genes, is becoming more important for the subsequent functional genomics studies. The newly optimized Red/ET recombination system was applied to the BAC modification. Mediated by the plasmid, pSC101-BAD-gbaA, and assisted by the counter-selectable/selectable system conferred by rpsL-Neo, two BACs were successfully modified. One step selectable BAC modification such as simple deletion or insertion was achieved in one week. For insertion or fusion of unselectable gene such as Cre, EFGP and LacZ as well as point mutation into targeted BACs, two rounds of Red/ET recombination was proceeded and two weeks was needed. After L-arabinose induction for transient expression of redy/red beta/red alpha/recA, the pSC101-BAD-gbaA plasmid died out spontaneously from the BAC host bacteria by shifting the temperature from 30 degrees C to 37 degrees C. Thus, there was no DNA contamination in the modified BACs being used for subsequent transgenesis research. The high efficient BAC modification mediated by optimized Red/ET recombination offers a significant facility to the functional genomics investigations.