Recombineering

Recombineering (recominogenic engbineering) is a genetic and molecular biology technique based on homologous recombination systems in E. coli to modify DNA. The term was first coined in 2001 by Ellis et al (Proc. Natl. Acad. Sci, 98:6742-6746, see page 6745) and reviewed by Copeland et al (Nature Genet. Rev., 2001, 2:769-779) Recombineering has been most successful using the bacteriophage lambda Red recombination system and the Rac-encoded RecET system. These homologous recombination systems mediate the efficient recombination of a target fragment (with homology sequences as short as 30 bps) into the DNA construct. The sequence homologies (or arms) flanking the desired modifications are homologous to regions 5' and 3' to the region to be modified. Positive and negative selections might be employed to increase the efficiency of this process.

Selection/Counterselection Technique
In the first stage of recombineering, a selection marker on a cassette is introduced to replace the region to be modified. In the second stage, a second counterselection marker (eg sacB) on the cassette is selected against following introduction of a target fragment containing the desired modification. Alternatively, the target fragment could be flanked by loxP or FRT sites, which could be removed later simply by the expression of the Cre or FLP recombinases, respectively.

Benefits
The biggest advantage of recombineering is that it obviates the need for conveniently positioned restriction sites, whereas in conventional genetic engineering, DNA modification is often compromised by the availability of unique restriction sites. In engineerig large constructs of >100 kb, such as the Bacterial Artificial Chromosomes (BACs), recombineering has become a necessity. Recombineering can generate the desired modifications without leaving any 'footprints' behind. It also forgoes multiple cloning stages for generating intermediate vectors and therefore is used to modify DNA constructs in a relatively short time-frame.

Recombineering