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Revision as of 16:34, 12 October 2018
Red/ET
The improvement of DNA recombination technologies has greatly enhanced the possibilities and convenience of mouse model systems usage. This improvement involves the construction of recombinant DNA molecules by recombineering in E.coli, the integration of this molecules into eucaryotic cells by homologous recombination or transposon techniques and the employment of site-specific recombinases to integrate recombinant DNA to alter those molecules.
What is Recombineering?
The original methods for the production of recombinant DNA along with oligonucleotide synthesis and PCR are limited by size due to their usage of restriction enzymes and DNA ligase to ‘‘cut-and-paste’’ DNA. However, recombineering is a revolutionary method for DNA engineering using homologous recombination. Recombineering allows unlimited cloning, subcloning, and modification of DNA at any chosen position. It permits precise engineering of DNA molecules of any size, including very large ones such as BACs or the E.coli chromosome.
Advantages over conventional methods:
•Independent of restriction sites
•No size limits
•No unwanted mutations
•Rapid
Recombineering was developed in A. Francis Stewart's Laboratory at the EMBL. This technical breakthrough is precise and independent of the presence of restriction sites and the size of the DNA molecule to be modified. Therefore, problems previously incurred by traditional DNA engineering techniques.
Furthermore this innovative technology is significantly less laborious than traditional DNA manipulation techniques and therefore has large savings on time and cost.
In Red/ET Recombineering, also referred to as λ-mediated recombination, target DNA molecules are precisely altered by homologous recombination in strains of E.coli that express phage-derived protein pairs, either RecE/RecT from the Racprophage, or Redα/Redβ from the λ phage. These protein pairs are functionally and operationally equivalent. RecE and Redα are 5´->3´ exonucleases, and RecT and Redβ are DNA annealing proteins. A functional interaction between RecE and RecT, or between Redα and Redβ is also required in order to catalyse the homologous recombination reaction.
Since the sequence of the homology regions can be chosen freely, any position on a target molecule can be specifically altered. Homologous recombination allows the exchange of genetic information between two DNA molecules in a precise, specific and faithful manner, qualities that are optimal for DNA engineering regardless of size.
One of the biggest advantages of recombineering is that it is not just limited to its unique ability to clone and subclone large DNA molecules but also it presents a variety of new ways to simplify conventional DNA engineering exercises . Recombineering allows every type of DNA modification possible. It becomes trivial to direct changes to a chosen DNA sequence or to introduce point mutations at any chosen site of a target DNA molecule regardless of its size.
The potential to use homologous recombination for DNA engineering had long been recognised, and methods which harnessed the endogenous homologous recombination system (termed RecA-dependent recombination) in E.coli, the premier cloning host, were developed . However this endogenous system has many limitations. It is impossible to use linear DNA molecules as they become rapidly degraded. Furthermore, recombination of circular molecules require relatively long homology regions and the ratio of intended to unwanted recombination products is extremely low. None of these limitations are encountered with recombineering.
How does Recombineering work?
Recombination occurs through homology regions, which are stretches of DNA shared by the two molecules that recombine. The recombination is further assisted by -encoded Gam protein, which inhibits the RecBCDexonuclease activity of E.coli. Unlike in yeast, linear dsDNA is unstable in E.coli because of the activity of RecBCD.
A double-stranded break repair (DSBR) is initiated by the recombinase protein pairs, RecE/RecT or Reda/Redb. First Red (or RecE) digests one strand of the DNA from the DSB, leaving the other strand as a 3’ ended, single-stranded DNA overhang. Then Red (or RecT) binds and coats the single strand. The protein-nucleic acid filament aligns with homologous DNA. Once aligned, the 3’ end becomes a primer for DNA replication.
Expression of Proteins for Recombineering
The λ-recombination functions can be expressed from a defective prophage integrated into the E.coli chromosome or from a plasmid. In the latter case Red/ET recombination is transferable to the host strain in which the BAC resides, thereby alleviating the need to retransform the BAC into a special strain.
One plasmid will be used in this course. The plasmid is carrying the red , , genes of the phage together with the recA gene in a polycistronic operon under the control of an inducible BAD-promoter. The pBAD promoter is both positively and negatively regulated by the product of the araC gene. AraC is a transcriptional regulator that forms a complex with L-arabinose. The recombination window is therefore limited by the transient nature of the induced expression of Red and Red, which are both strictly required for Red/ET recombination to occur. Thus, the risk of unwanted intramolecular rearrangement is minimized, allowing recombinants that contain no other unintended changes to be recovered efficiently.
While constitutive expression of the red gene has a toxic effect in DH10B (recA-) cells under some conditions, thus limiting the efficiency of recombination, tightly regulated expression of the gene together with simultaneous expression of the Red and Red genes allows efficient homologous recombination between linear DNA fragments and BACs resident in such cells as DH10B.
The plasmid is a derivative of the temperature-sensitive pSC101 replicon which is a low copy number plasmid, depending on the oriR101. The RepA protein encoded by plasmid pSC101 is required for plasmid DNA replication and the partitioning of plasmids to daughter cells at divisions. Since a temperature-sensitive (Ts) RepA protein is expressed, cells have to be cultured at 30C, pSC101 derivatives are easily curable at 37C to 43C.
Experiments have shown that after 2h of cell growth at a temperature non-permissive for replication of this plasmid (i.e. 42C), the average plasmid copy number is sharply deceased by about 80% during four generations of bacterial cell growth at 42C. After return of the cultures to 30C, approximately the same number of generations of bacterial cell growth is required for the copy number of the plasmids to return to the level observed before.
Since the plasmid is depending on the oriR101 it can be propagated in E.coli together with most ColE1-derived plasmids. The temperature-sensitivity of the RepA proteins is leading to a rapid loss of the plasmid in the absence of continued selection pressure.
Applications
Recombineering can be applied to numerous DNA engineering tasks, including:
subcloning by gap repair, e.g. for subcloning from genomic DNA or BACs
insertion of selectable fragments or/and deletion, e.g. BAC modification;
oligonucleotide-directed mutagenesis
Those applications have also been successfully adapted to high-throughput DNA engineering methods.
Combined and sequential use of those applications enables a variety and modularity of DNA modifications. As an example we show our current method to make targeting constructs starting from a BAC.