Recombineering
Recombineering refers to recombination-mediated genetic engineering and specifies a well-defined and precise modification of DNA. This outstanding technology was developed by the group of A. Francis Stewart at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany in the late 1990s.
Gene Bridges’ Red/ET recombination technique is an in vivo reaction and is based either on the red genes of phage lambda or the recE/recT genes of the Rac prophage. The respective gene cluster is under the control of an artificial promoter system and hence inducible to express the recombination supporting enzymes in an E. coli host cell. Recombination between two homologues DNA molecules is mediated by the functional interaction of the two corresponding protein partners, whereby Redα or RecE are acting as a 5’→3’ exonuclease and Redβ or RecT as DNA single strand binding protein. At least one of the DNA molecules has to be linear with analogous homology regions on both sides.
Most importantly only 50 bp homology arms allow nucleotide-precise DNA modification at any desired position without limitation to restriction sites, since the sequence of the homology regions can be chosen freely. Furthermore DNA engineering may be performed using DNA stretches ranging from short oligonucleotides to large fragments of 10 kb or oven larger. Consequently the recombineering is suitable for a wide range of application such as subcloning from large DNA molecules (BACs, bacterial chromosomes) into plasmid backbones, insertion or deletion of DNA fragments and introducing of point mutations into open reading frames.
The Red/ET technology is an elaborate tool to address a diverse spectrum of scientific questions, for example for the creation of tailor-made constructs for the generation of animal models and any possible BAC-modifications, as well as for modifications of chromosomes, e.g. for pathway engineering.
To obtain an impression please have a closer look at the diverse examples generated by the Gene Bridges engineering team:
Examples for constructs for animal models and BAC modifications :
Construct to obtain a mouse model for the human disease “Seckel syndrome”
Obtain a new animal model for Huntington’s Disease
Construction of chimeric human-rat IgH loci by BAC stitching and counterselection