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.