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PCR samples were run in 1% agarose gels with TBE (45 mM Tris–base, 45 mM boric acid, 10 mM EDTA, pH cool for 1.5 h at 80 V (60 mA). Gels were stained with ethidium bromide. Image recording was performed with a Chemidoc EQ BioRad photodocumenter.
Reverse PCR with Δ202 genomic DNA. Since the Δ202 construct has no XbaI restriction sites, rPCR was performed with DNA from affected Δ202 mice digested with XbaI to generate linear fragments circularized and sealed with ligase. Circular DNA LAQ824 containing the transgene flanked by mouse genome sequences would serve as templates to generate products containing transgene and mouse genomic sequences from which the insertion site could be identified.
Four-μg DNA from Δ202 mice were digested 3 h with 1 U of XbaI at 37 °C. Circular molecules were sealed by overnight incubation at 16 °C with 1 U of T4 ligase. Three successive rPCR rounds were carried out with primer sets 3, 4, and 5, which bind to sites progressively closer to the transgene ends (Table 1 and Fig. 1B).