Fig 1.
Different types of recombineering processes.
(A) Insertional recombination. A selection marker (sm) containing homology to a target site is inserted during the process of DNA replication (dashed line). Gap repair cloning. A gapped plasmid with terminal homology regions to a target site is used to subclone a sequence of interest. (C) Subcloning plus insertion (SPI). Insertion of a cassette occurs simultaneously during subcloning and generates a targeted subcloned plasmid. The template DNA remains unmodified.
Fig 2.
Effect of homology length on singleplex and multiplex recombination.
(A) Insertion assay. A Gentamicin lagging strand protected cassette was PCR generated with different homology lengths (20 bp, 35, bp, 60 bp, 90 bp, 120, bp and 180 bp) and inserted at a site of the mouse P2rx1 gene. Data points represent averages; error bars indicate standard error of mean (n = 3). The recombination frequencies plotted here and in the subsequent figures are shown in S1 Table. (B) Gap repair assay. Gap repair was performed at the P2rx1 locus using p15A zeo lagging strand protected subcloning plasmids containing different HLs. Gap repair frequency was calculated using PCR genotyping of 24 clones for each sample (n = 3). The 20 bp homology did not yield any correct gap repaired plasmids. (C) Multiplex insertion assay. Homology series of two different Zeocin and Gentamicin lagging strand protected cassettes both containing the same HL were PCR generated and simultaneously inserted at two different sites of the P2rx1 gene (n = 3). (D) SPI assay. A SPI assay was performed at the P2rx1 gene using a p15A zeo lagging strand protected subcloning plasmid containing 230 bp homology regions and the Gentamicin cassettes used in (A) (n = 3). (E) PCR analysis of multiplex insertion assay. Recombinants were genotyped with an insertion cassette specific primer and a homology region flanking primer. Positive clones were used to isolate BAC DNA and long range PCR was performed at each of the insertion sites using both homology region flanking primers. The presence of the wild-type (wt) band in a sample indicates the presence of mixtures of BAC plasmids denoted by a star symbol. T, P2rx1 zeo genta BAC (positive control); w, P2rx1 wild-type BAC (negative control); M, 1 kb+ ladder (Invitrogen). (F) Restriction enzyme (RE) analysis of SPI assay with one cassette. Plasmid DNA was digested with EcorV and SspI. Diamond symbol denotes samples containing targeted (T) and non-targeted (w) p15A plasmids. (G) RE analysis of SPI assay with two insertion cassettes. SPI was performed using a p15A dhfrII lagging strand protected subcloning plasmid containing 230 bp homology regions and Zeocin and Gentamicin lagging strand protected cassettes containing 35 bp or 60 bp homologies. Clones were analysed with KpnI.
Fig 3.
Multiplex recombination is limited by availability of DNA template.
(A) Insertion assay. A Gentamicin lagging strand protected cassette was inserted at a site of the P2rx1 gene. (B) Multiplex insertion. Two different Gentamicin and Zeocin lagging strand protected cassettes were inserted at two different sites of the P2rx1 gene. Recombination assays were performed with different amounts of each DNA cassette in the electroporation. Values represent averages; error bars indicate standard error of mean (n = 4). (C) Multiplex insertion PTO assay. Three different Zeocin, Gentamicin and Blasticidin resistance cassettes were PTO protected or unmodified and inserted at three different sites of the P2rx1 gene (n = 3). (D) SPI PTO assay. SPI was performed using a p15A dhfrII subcloning plasmid and the Zeocin and Gentamicin cassettes used in (A) (n = 3).
Fig 4.
Co-selection is required for efficient multiplex recombination.
Multiplex insertion was performed using a pBeloBAC11 plasmid (19.5 kb total size) containing the region of the P2rx1 gene shown in S1 Fig. The Zeocin and Gentamicin lagging strand protected cassettes were co-transformed with a Kanamycin resistance plasmid and selected with Zeocin and Kanamycin or Kanamycin only. BAC DNA was prepared using the QIAquick Spin Miniprep kit (Qiagen) from each selection scheme and 100 ng of DNA was transformed into HS996 cells and plated with combined Zeocin and Gentamicin selection. Values represent averages; error bars indicate standard error of mean (n = 3).
Fig 5.
Requirement of different Red proteins for singleplex and multiplex recombination.
(A) Insertion. A Gentamicin resistance cassette was inserted at a site of the P2rx1 gene. (B) Gap repair. A p15A zeo subcloning plasmid was used to subclone a region of the P2rx1 gene. (C) Multiplex insertion. Two different Gentamicin and Zeocin resistance cassettes were inserted at two different sites of the P2rx1 gene. (D) SPI assay. The p15A zeo subcloning plasmid was used together with the Gentamicin resistance cassette. Recombination assays were performed using lagging strand protected cassettes and with expression of different Red proteins. Values represent average; error bars indicate standard error of mean (n = 3 for A, B and D, n = 4 for C).
Fig 6.
Multiplex recombination is increased in an Exo VII deletion strain.
(A) Multiplex insertion. (B) SPI. Three different Gentamicin, Zeocin and Blasticidin lagging strand protected cassettes were inserted at three different sites of the P2rx1 gene: 1 cassette, Gentamicin; 2 cassettes, Gentamicin and Zeocin; 3 cassettes, Gentamicin, Zeocin and Blasticidin. The p15A dhfrII lagging strand protected subcloning plasmid was used in the SPI assays. Data points represent averages; error bars indicate standard error of mean (n = 3 for A and B).
Table 1.
Comparision of parameters affecting multiplex recombination of oligos and dsDNA cassettes.