**2. Overview of recombineering technology**

Traditional cloning approaches rely on the presence of unique restriction enzyme sites for modification of plasmid DNA via a series of digestions and ligations to incorporate or remove desired DNA sequences. Unfortunately, most restriction enzyme sequences are not unique or conveniently located within the genomic sequence. Thus, the availability/use of restriction sites is often a limiting factor when attempting to modify plasmid DNA using such approaches.

Recombineering technology achieves DNA modification using a phage homologous recombination system, which uses linear DNA as template. Thus, an investigator can use linear targeting vectors containing 5' and 3' arms with homology to a target locus to introduce new DNA sequence. Subtle changes can now be achieved, including single point mutations. Since the modification to the locus is based solely on sequence present in the target and not restriction enzymes, DNA can be introduced to the target wherever needed. Thus, recombineering technology has opened up an unlimited number of possibilities for genetic modifications.

Prior to the use of lambda phage in Recombineering, the study of homologous recombination in E. coli laid the groundwork for the use of this technology. Homologous recombination via linear DNA is suppressed in E. coli by the recBCD enzyme complex. In the recBCD model, the enzyme complex moves destructively along double strand breaks. The recB and recC subunits operate as helicases while recD operates as an exonuclease. Thus, in recBCD wild type E. coli strains, recombination does not occur through linear DNA, as reviewed elsewhere (Myers and Stahl 1994; Yeeles and Dillingham 2010).

The recBCD complex moves destructively along linear DNA until it encounters a DNA motif called a chi site. The chi site motif is a "recombination hotspot" that facilitates homologous recombination by ejecting the recD subunit responsible for the exonuclease activity of the complex, but does not affect the helicase activity. The helicase activity results in single strand DNA that serves as substrate for homologous recombination. This molecular reaction was exploited by incorporation of chi sites into targeting vectors to enable homologous recombination of target genes.

Cloning by homologous recombination was also studied in recBCD deficient strains. These experiments used recBCD mutants in an attempt to modify bacterial chromosomal and plasmid DNA with a linear DNA targeting construct (Jasin and Schimmel 1984; Oliner, Kinzler et al. 1993). The success of these studies demonstrated the effectiveness of cloning by homologous recombination, but was dependent on the use of specialized bacterial strains with constitutively active recombination enzymes. This enzyme activity resulted in unwanted intramolecular rearrangements in the modified plasmid (Copeland, Jenkins et al. 2001). Therefore, the recBCD mutant strains were limited in their use in cloning by homologous recombination.

The Recombineering technology used today is based on the lambda phage Red double strand break repair system, which uses the phage proteins *exo*, *bet*, and *gam*. This system is initiated when the 5'to3' exonuclease *exo* digests linear double stranded (ds) DNA leaving a 3' overhang of single stranded (ss) DNA. The resultant 3'ssDNA is coated by the *bet* protein, which facilitates its annealing to a complementary strand of DNA. Once the homologous DNA is annealed, the 3'OH becomes a priming site for DNA replication resulting in double strand break repair.

The activities of *exo*, *bet*, and *gam* have been adapted for BAC cloning by homologous recombination. The dsDNA substrate for *exo* is a linear targeting construct with 5' and 3' homology to the target locus. The linear targeting construct can be generated by PCR or excised from a plasmid. The resultant 3'ssDNA is coated by the *bet* protein and facilitates the annealing of the 3'ssDNA of the targeting construct to the targeted sequence on the BAC containing the gene locus. The linear targeting construct is unaffected by recBCD activity due to the presence of the *gam* protein, which inhibits recBCD binding to the dsDNA targeting construct (Murphy 2007).

Studies that employed the introduction of lambda phage Red double strand break repair into E. coli demonstrated that it was an efficient system for cloning by homologous

mutations. Since the modification to the locus is based solely on sequence present in the target and not restriction enzymes, DNA can be introduced to the target wherever needed. Thus, recombineering technology has opened up an unlimited number of possibilities for

Prior to the use of lambda phage in Recombineering, the study of homologous recombination in E. coli laid the groundwork for the use of this technology. Homologous recombination via linear DNA is suppressed in E. coli by the recBCD enzyme complex. In the recBCD model, the enzyme complex moves destructively along double strand breaks. The recB and recC subunits operate as helicases while recD operates as an exonuclease. Thus, in recBCD wild type E. coli strains, recombination does not occur through linear

The recBCD complex moves destructively along linear DNA until it encounters a DNA motif called a chi site. The chi site motif is a "recombination hotspot" that facilitates homologous recombination by ejecting the recD subunit responsible for the exonuclease activity of the complex, but does not affect the helicase activity. The helicase activity results in single strand DNA that serves as substrate for homologous recombination. This molecular reaction was exploited by incorporation of chi sites into targeting vectors to

Cloning by homologous recombination was also studied in recBCD deficient strains. These experiments used recBCD mutants in an attempt to modify bacterial chromosomal and plasmid DNA with a linear DNA targeting construct (Jasin and Schimmel 1984; Oliner, Kinzler et al. 1993). The success of these studies demonstrated the effectiveness of cloning by homologous recombination, but was dependent on the use of specialized bacterial strains with constitutively active recombination enzymes. This enzyme activity resulted in unwanted intramolecular rearrangements in the modified plasmid (Copeland, Jenkins et al. 2001). Therefore, the recBCD mutant strains were limited in their use in cloning by

The Recombineering technology used today is based on the lambda phage Red double strand break repair system, which uses the phage proteins *exo*, *bet*, and *gam*. This system is initiated when the 5'to3' exonuclease *exo* digests linear double stranded (ds) DNA leaving a 3' overhang of single stranded (ss) DNA. The resultant 3'ssDNA is coated by the *bet* protein, which facilitates its annealing to a complementary strand of DNA. Once the homologous DNA is annealed, the 3'OH becomes a priming site for DNA replication resulting in double

The activities of *exo*, *bet*, and *gam* have been adapted for BAC cloning by homologous recombination. The dsDNA substrate for *exo* is a linear targeting construct with 5' and 3' homology to the target locus. The linear targeting construct can be generated by PCR or excised from a plasmid. The resultant 3'ssDNA is coated by the *bet* protein and facilitates the annealing of the 3'ssDNA of the targeting construct to the targeted sequence on the BAC containing the gene locus. The linear targeting construct is unaffected by recBCD activity due to the presence of the *gam* protein, which inhibits recBCD binding to the dsDNA

Studies that employed the introduction of lambda phage Red double strand break repair into E. coli demonstrated that it was an efficient system for cloning by homologous

DNA, as reviewed elsewhere (Myers and Stahl 1994; Yeeles and Dillingham 2010).

enable homologous recombination of target genes.

homologous recombination.

strand break repair.

targeting construct (Murphy 2007).

genetic modifications.

recombination (Murphy 1998). Other studies demonstrated the use of homology arms as short as 27nt in length facilitated cloning by homologous recombination, with increasing efficiency with increased homology length. This study also investigated insert length between homologous arms and found it useful for inserts from 0-3100bp in length (Zhang, Buchholz et al. 1998). It was with these findings that lambda phage Red double strand break repair was adopted for cloning by homologous recombination.
