Chromosome Engineering by Homologous Recombination

最近，我们开发了一种基于lambda介导的同源重组(Red)的体内克隆和基因修饰系统。这个系统消除了用限制性内切酶切割DNA或用DNA连接酶连接DNA片段的需要。我们相信，这种极其高效的重组系统将彻底改变重组DNA的工作方式。单链DNA寡核苷酸(短至20个碱基)和线性双链DNA(短至30个碱基)均可用于在细菌染色体、质粒或基因组BAC文库克隆上产生重组子。单链DNA寡核苷酸已被用于创建点突变、修复突变、创建缺失以及在染色体和BAC克隆中创建小插入。单链DNA的重组只需要Lambda噬菌体Beta蛋白，它与电穿孔进入细胞的单链DNA寡核苷酸结合，并使该核苷酸与其在细胞中的目标同源性相结合。在复制过程中形成的单链DNA中间体发生退火。重组长dsDNA(&gt；1000bp)除了需要Beta外，还需要Lambda噬菌体功能Exo和Gam。Exo降解dsDNA的5‘末端，产生供Beta结合的单链DNA突出物。GAM抑制宿主核酸酶破坏进入的线性DNA。通过这种体内重组工程技术，各种细菌载体已经被开发出来，以确保高效和准确的基因工程。

Recently, we have developed an in vivo cloning and gene modification system using lambda-mediated homologous recombination (Red). This system eliminates the need to cut DNA with restriction enzymes or join DNA fragments with DNA ligase. This extremely efficient recombination system will we believe, revolutionize the way in which recombinant DNA work is done. Both ssDNA oligos (as short as 20 bases) and linear dsDNA (as short as 30 bp) can be used to generate recombinants on the bacterial chromosome, on plasmids, or on genomic BAC library clones. ssDNA oligos have been used to create point mutations, to repair mutations, to create deletions, and to create small insertions on the chromosome and in BAC clones. Recombination of ssDNA requires only the lambda phage Beta protein which binds the ssDNA oligonucleotide that is electroporated into cells and anneals that nucleotide to its target homology in the cell. Annealing occurs to the ssDNA intermediates that form during replication. Recombination of long dsDNA (>1000bp) requires lambda phage functions Exo and Gam in addition to Beta. Exo degrades 5' ends of dsDNA generating ssDNA overhangs for Beta to bind. Gam inhibits host nucleases which destroy the entering linear DNA. Various bacterial vectors have been developed to ensure efficient and accurate genetic engineering via this in vivo recombineering technology.