Definition of Recombineering

Recombineering (recombination-mediated genetic engineering) is a genetic and molecular biology

Molecular biology

Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 technique based on homologous recombination

Homologous recombination

Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...

 systems, as opposed to the older/more common method of using restriction enzymes and ligase

Ligase

In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...

s to cut and glue DNA sequences. It has been developed in E. coli and now is expanding to other bacteria species and is used to modify DNA in a precise and simple manner. The procedure is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout

Knockout mouse

A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA...

, and for modifying DNA of any source often contained on a bacterial artificial chromosome

Bacterial artificial chromosome

A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...

 (BAC).

Early history of the techniques

Although developed in bacteria, much of the inspiration for recombineering techniques came from methods first developed in Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...

  where a linear plasmid was used to target genes or clone genes off the chromosome. In addition, recombination with single-strand oligonucleotides (oligos) was first shown in Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...

. They saw recombination with oligonucleotides as short as 20 bases.

Recombineering is based on homologous recombination in Escherichia coli mediated by phage proteins, either RecE/RecT from Rac prophage or Redαβδ from bacteriophage lambda. The lambda Red recombination system is now most commonly used and the first demonstrations of Red in vivo genetic engineering were independently made by Kenan Murphy and Francis Stewart . However, Murphy's experiments required expression of RecA and also employed long homology arms. Consequently the implications for a new DNA engineering technology were not obvious. The Stewart lab showed that these homologous recombination systems mediate efficient recombination of linear DNA molecules flanked by homology sequences as short as 30 bps (40-50 bps are more efficient) into target DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 sequences in the absence of RecA. Now the homology could be provided by oligonucleotides made to order, and standard recA cloning hosts could be used, greatly expanding the utility of recombineering.

Recombineering with dsDNA

Recombineering utilizes linear DNA substrates that are either double-stranded (dsDNA) or single-stranded (ssDNA). Most commonly, dsDNA recombineering has been used to create gene replacements, deletions, insertions, inversions. Gene cloning and gene/protein tagging (His tags etc., see ) is also common. For gene replacements or deletions, usually a cassette encoding a drug-resistance gene is made by PCR using bi-partite primers. These primers consist of (from 5’→3’) 50 bases of homology to the target region, where the cassette is to be inserted, followed by 20 bases to prime the drug resistant cassette. The exact junction sequence of the final construct is determined by primer design. These events typically occur at a frequency of approximately 10⁴/10⁸cells that survive electroporation

Electroporation

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...

. Electroporation is the method used to transform the linear substrate into the recombining cell.

Selection/Counterselection Technique

In some cases, one desires a deletion with no marker left behind, to make a gene fusion, or to make a point mutant in a gene. This can be done with two rounds of recombination. In the first stage of recombineering, a selection marker on a cassette is introduced to replace the region to be modified. In the second stage, a second counterselection marker (e.g. sacB) on the cassette is selected against following introduction of a target fragment containing the desired modification. Alternatively, the target fragment could be flanked by loxP

Cre-Lox recombination

Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice . Subsequently, the laboratory of Dr...

or FRT

FLP-FRT Recombination

In genetics, FLP-FRT recombination is a site-directed recombination technology used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-Lox recombination...

sites, which could be removed later simply by the expression of the Cre

Cre recombinase

Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction...

 or FLP recombinases, respectively.

Recombineering with ssDNA

Recombineering with ssDNA provided a breakthrough both in the efficiency of the reaction and the ease of making point mutations. This technique was further enhanced by the discovery that by avoiding the methyl-directed mismatch repair system, the frequency of obtaining recombinants can be increased to over 10⁷/10⁸ viable cells. This frequency is high enough that alterations can now be made without selection. With optimized protocols, over 50% of the cells that survive electroporation contain the desired change. Recombineering with ssDNA only requires the Red Beta protein; Exo, Gamma and the host recombination proteins are not required. As proteins homologous to Beta and RecT are found in many bacteria and bacteriophages (>100 as of February 2010), recombineering is likely to work in many different bacteria. Thus, recombineering with ssDNA is expanding the genetic tools available for research in a variety of organisms. To date, recombineering has been performed in E. coli, S. enterica, Y. pseudotuberculosis, and M. tuberculosis.

Red-Independent recombination

Recently, it has been demonstrated that ssDNA recombination can occur in the absence of known recombination functions. Recombinants were found at up to 10⁴/10⁸ viable cells. This Red-independent activity has been demonstrated in P. syringae, E. coli, S. enterica serovar typhimurium and S. flexneria.

Benefits of recombineering vs other genetic engineering techniques

The biggest advantage of recombineering is that it obviates the need for conveniently positioned restriction sites

Restriction enzyme

A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...

, whereas in conventional genetic engineering, DNA modification is often compromised by the availability of unique restriction sites. In engineering large constructs of >100 kb, such as the Bacterial Artificial Chromosome

Bacterial artificial chromosome

A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...

s (BACs), or chromosomes, recombineering has become a necessity. Recombineering can generate the desired modifications without leaving any 'footprints' behind. It also forgoes multiple cloning

Cloning

Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...

 stages for generating intermediate vectors and therefore is used to modify DNA constructs in a relatively short time-frame. The homology required is short enough that it can be generated in synthetic oligonucleotides and recombination with short oligonucleotides themselves is incredibly efficient. Recently, recombineering has been developed for high throughput DNA engineering applications termed 'recombineering pipelines'. Recombineering pipelines support the large scale production of BAC transgenes and gene targeting

Gene targeting

Gene targeting is a genetic technique that uses homologous recombination to change an endogenous gene. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Gene targeting can be permanent or conditional...

constructs for functional genomics programs such as EUCOMM (European Conditional Mouse Mutagenesis Consortium) and KOMP (Knock-Out Mouse Program). Recombineering has also been automated, a process called "MAGE" -Multiplex Automated Genome Engineering, in the Church lab.

External links

• http://redrecombineering.ncifcrf.gov/ This website contains more details about recombineering as well as protocols, FAQ's and can be used to request strains and plasmids needed for recombineering.