Difference between revisions of "Team:SKLMT-China/Red ET"

introductin

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 (Cohen et al., 1973; Mullis et al., 1986) have come to an end (Zhang et al., 1998; Zhang et al., 2000) . Furthermore this innovative technology is significantly less laborious than traditional DNA manipulation techniques and therefore has large savings on time and cost (Muyerset al., 1999) . In Red/ET Recombineering, also referRed to as \({\lambda}\)-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 Red\({\alpha}\)/Red\({\beta}\) from the \({\lambda}\) phage (Muyerset al., 2000). These protein pairs are functionally and operationally equivalent. RecE and Red\({\alpha}\) are 5´-3´ exonucleases, and RecT and Red\({\beta}\) are DNA annealing proteins. A functional interaction between RecE and RecT, or between Red\({\alpha}\) and Red\({\beta}\) 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 (Muyerset al., 1999; Muyerset al., 2000). 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 [6]. 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 γ-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 Red\({\alpha}\)/Red\({\beta}\). First Red\({\alpha}\) (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\\({\beta}\)(or RecT) binds and coats the single strand (Erleret al., 2009). The protein-nucleic acid filament aligns with homologous DNA. Once aligned, the 3’ end becomes a primer for DNA replication (Muyerset al., 2000; Marescaet al., 2010).

Expression of Proteins for Recombineering

The \({\gamma}\)-recombination functions can be expressed from a defective prophage integrated into the E.coli chromosome (e.g. Zhang et al. 2000) or from a plasmid (see below). In the latter case Red recombination is transferable to the host strain in which the BAC resides, thereby alleviating the need to retransform the BAC into a special strain.