Recombineering (recombination-mediated genetic engineering) is a powerful method
for fast and efficient construction of vectors for subsequent manipulation of the
mouse genome or for use in cell culture experiments. It is also an efficient way
of manipulating the bacterial genome directly.
Recombineering is a method based on homologous recombination in E. Coli using recombination
proteins provided from λ phage.
Our bacterial strains contain a defective λ prophage inserted into the bacterial
genome. The phage genes of interest, exo, bet, and gam, are transcribed from the
λPL promoter. This promoter is repressed by the temperature-sensitive repressor
cI857 at 32°C and derepressed (the repressor is inactive) at 42°C. When bacteria
containing this prophage are kept at 32°C no recombination proteins are produced.
However, after a brief (15 minutes) heat-shock at 42°C a sufficient amount of recombination
proteins are produced. exo is a 5'-3' exonuclease that creates single-stranded overhangs
on introduced linear DNA. bet protects these overhangs and assists in the subsequent
recombination process. gam prevents degradation of linear DNA by inhibiting E. Coli
Linear DNA (PCR product, oligo, etc.) with sufficient homology in the 5' and 3'
ends to a target DNA molecule already present in the bacteria (plasmid, BAC, or
the bacterial genome itself) can be introduced into heat-shocked and electrocompetent
bacteria using electroporation. The introduced DNA will now be modified by exo and
bet and undergo homologous recombination with the target molecule. The method is
so efficient that co-electroporation of a supercoiled plasmid and a linear piece
of DNA into heat-shocked, electrocompetent bacteria will work as well.
For additional information on protocols, strains, and recombineering agents being
developed by the Court lab at NCI-Frederick please check their website: http://redrecombineering.ncifcrf.gov/