Recombineering

Recombineering (recombination-mediated genetic engineering) is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.

Development

Although developed in bacteria, much of the inspiration for recombineering techniques came from methods first developed in Saccharomyces cerevisiae where a linear plasmid was used to target genes or clone genes off the chromosome. In addition, recombination with single-strand oligonucleotides (oligos) was first shown in Saccharomyces cerevisiae. They saw recombination with oligonucleotides as short as 20 bases.

Recombineering is based on homologous recombination in Escherichia coli mediated by bacteriophage proteins, either RecE/RecT from Rac prophage or Redαβδ from bacteriophage lambda. The lambda Red recombination system is now most commonly used and the first demonstrations of Red in vivo genetic engineering were independently made by Kenan Murphy and Francis Stewart. However, Murphy's experiments required expression of RecA and also employed long homology arms. Consequently the implications for a new DNA engineering technology were not obvious. The Stewart lab showed that these homologous recombination systems mediate efficient recombination of linear DNA molecules flanked by homology sequences as short as 30 base pairs (40-50 base pairs are more efficient) into target DNA sequences in the absence of RecA. Now the homology could be provided by oligonucleotides made to order, and standard recA cloning hosts could be used, greatly expanding the utility of recombineering.