一种基于Red的在大肠杆菌中修饰染色体和BAC的新型重组工程系统

A new Red-based recombineering system used for modifying chromosome and BACs in Escherichia coli

近 5年来兴起了一种新型的基于λ 噬菌体Red重组系统的重组工程技术 ,它将缺陷型λ 噬菌体整合入大肠杆菌染色体上 ,使 3种重组酶蛋白Exo、Beta和Gam的表达受到严格的调控。Red重组工程技术不受酶切位点的限制 ,只需用 35～ 5 0bp长的同源臂就能得到很高的重组效率。运用这种技术能在大肠杆菌体内通过同源重组对大肠杆菌染色体、BAC质粒等复杂DNA片段上的基因进行突变、敲除、替换等修饰 ;此外可运用该技术中缺口修复的方法构建质粒。Red重组工程技术有远大的应用前景 ,将极大地推动功能基因组研究的进展。

In the past five years, a new Red re combination system-based recombineering technology hasbeen developed. I twas made by integratinga defective λprophage into the Escherichia coli chromosome. Thedefective λphage harbors th ese recombination genes whoseexpressionsare tightly controlled. All that the efficient prophage recombination system requires is the synthesis o f standard oligonucleotides or construction of PCR products that provide the hom ologous sequences as short as 35 to 50 base pairs without the need for restricti on enzymes.This technique has been used to modify the genes of E.coli chromosome and BAC plasmid for mutation, knock-out, substitutionand so on. In addition,constructed plasmids can be by gap repair of this technology. R ed recombineering is veryuseful and will promote the advance in function al genomics.