Recombineering

Recombineering (recombinogenic engineering) is a genetic and molecular biology technique based on homologous recombination systems in E. coli to modify DNA. The procedure is widely used in the generation of target vectors for making conditional mouse knockout. The term was first coined in 2001 by Ellis et al (Proc. Natl. Acad. Sci, 98:6742-6746, see page 6745) and reviewed by Muyrers et al (Trends Biochem Sci. 2001, 26, 325-3), Copeland et al (Nature Genet. Rev., 2001, 2:769-779), Court et al, (Ann Rev Genetics 2002, 36, 361-88). Recombineering is based on homologous recombination in Escherichia coli mediated by phage proteins, either RecE/RecT from Rac prophage or Red alpha/beta/gamma from bacteriophage lambda (Zhang et al, Nature Genetics, 1998, 20, 123-8; Muyrers et al, Nucleic Acids Res., 1999, 27, 1555-7). The lambda Red recombination system is now most commonly used. 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.

election/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 engineering 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. Recently, recombineering has been developed for high throughput DNA engineering applications termed 'recombineering pipelines' (Sarov et al, Nature Methods 2006, 3, 839-44). Recombineering pipelines support the large scale production of BAC transgenes and gene targeting constructs for functional genomics programs such as EUCOMM (European Conditional Mouse Mutagenesis Consortium) and KOMP (Knock-Out Mouse Program).