Genetic engineering has traditionally been done in Escherichia coli using restriction enzymes to cleave DNA, and using DNA ligases to join them. However, there are several limitations to this approach, especially when large DNA molecules require engineering, because even rare restriction enzymes occur over large stretches of DNA. The generation of transgenic and mouse knockout constructs in E. coli is also hampered by the difficulty of finding appropriately placed restriction-enzyme cleavage sites.
Genetic engineering in yeast alleviates these problems because it relies on homologous recombination rather than on restriction enzymes and DNA ligases to generate recombinant DNA molecules.
A principal limitation of genetic engineering in yeast is that yeast artificial cloning vectors (YACs), developed for cloning large DNA molecules, are often unstable, and YAC transgenic mice are difficult to make. As a result, more stable vectors, such as bacterial artificial chromosomes (BACs), are often used instead.
Phage-based E. coli recombination systems have been developed that now allow large DNA molecules cloned into BACs to be modified by homologous recombination, similarly to what occurs in yeast. These E. coli recombination systems have many of the advantages of yeast recombination, but few of its disadvantages.
This new form of chromosome engineering, termed recombineering, makes it possible to introduce virtually any type of mutation into a BAC using PCR-amplified, linear, double-stranded DNA targeting cassettes that have short regions of homology at their ends, or single-stranded oligonucleotides.
Recombineering greatly decreases the time it takes to create transgenic mouse models by conventional means. It also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and it should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.
Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.
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- PHAGE-BASED RECOMBINATION
Bacteria, such as Escherichia coli, encode their own homologous recombination systems. Viruses or phages that inhabit bacteria also often carry their own recombination functions, which can work with, or independently of, the bacterial recombination functions.
- BACTERIAL ARTIFICIAL CHROMOSOME
(BAC). A cloning vector derived from a single-copy F-plasmid of Escherichia coli. It carries the F replication and partitioning systems that ensure low-copy number and faithful segregation of plasmid DNA to daughter cells. Large genomic fragments can be cloned into F-type plasmids, making them useful for constructing genomic libraries.
An autonomously replicating DNA that is often marked with a gene that encodes drug resistance, which allows selection for cells that carry the plasmid.
A yeast selectable marker that encodes an enzyme required for histidine (His) biosynthesis. HIS3 yeast mutants cannot grow in media without His. On HIS-deficient medium, recombinants that restore the wild-type gene are able to grow again.
- GAP REPAIR
A linear plasmid vector (gapped vector) can be circularized by homologous recombination between its ends and a target DNA.
- SHUTTLE VECTOR
A plasmid that can be moved from one species to another, such as plasmids that contain origins of replication for both yeast and bacterial hosts.
RecA is central to recombination in Escherichia coli, and all organisms have RecA homologues. It allows two homologous DNAs to find each other, and to trade DNA strands by binding to a single-stranded region in one of the DNAs and by using that strand to search for its double-stranded DNA (dsDNA) homologue. It then binds to a homologue, causing the single strand to pair with its complement in the dsDNA, displacing the identical strand of the duplex and generating a key intermediate in the recombination process.
- POSITIVE–NEGATIVE SELECTION
When the presence of a cassette is positively selected for, for example by drug resistance, and then negatively selected for, by eliminating cells that express a second selectable marker.
A strain of Escherichia coli that has been modified and selected to accept large BAC clones by transformation. DH10B is defective for RecA recombination.
- RAC PROPHAGE
Escherichia coli and other bacteria contain, in their chromosomes, remnants of viruses or prophages, such as Rac in E. coli, that often are defective and contain only a few genes of the original virus. Two Rac genes, recE and recT, encode homologous recombination functions, and are normally silent, but the sbcA mutation activates their constitutive expression.
A simple five-carbon sugar metabolized by Escherichia coli, which is used as a chemical to induce and activate expression from the promoter pBAD.
Cre is a site-specific recombinase that recognizes and binds to specific sites called loxP. Two loxP sites recombine at nearly 100% efficiency in the presence of Cre, allowing DNA cloned between two such sites to be removed by Cre-mediated recombination.
Flpe is a genetically enhanced, site-specific Flp recombinase that recognizes and binds to FRT sites.
A derivative of DH10B. The defective λ-prophage, used to express the red and gam functions, has been moved into the chromosome of this strain.
sacB encodes the SacB protein, which converts sucrose into a toxic form that kills bacteria. This can be used in negative selection for the sacB gene.
- FLAG TAG
A short peptide sequence that is added to a protein to allow the protein to be recognized by antibodies raised against the flag tag.
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Copeland, N., Jenkins, N. & Court, D. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2, 769–779 (2001). https://doi.org/10.1038/35093556
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