Mini-λ: a tractable system for chromosome and BAC engineering

Abstract

The bacteriophage lambda (λ) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective λ prophage. Here we describe the generation of a mini-λ DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E. coli strain, including the DH10B-carrying BACs or PACs. The mini-λ DNA integrates into the bacterial chromosome as a defective prophage. In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA. We describe here the use of the mini-λ recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone. In addition, using the mini-λ, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker. The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-λ as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering.