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. 2015 Aug 14;10(8):e0134672. doi: 10.1371/journal.pone.0134672

Fig 1. Generation of Rb1_PPP1R26P1 knock-in mice.

Fig 1

A) Top: Scheme of the targeting vector, consisting of two homology arms, a neomycin selection cassette flanked by loxP511 sites, and the PPP1R26P1 human pseudogene. Middle: the wildtype mouse Rb1 allele, with the site of integration shown. Bottom: the targeted and recombined allele. B) Southern blot of selected ES clones and tail snips of generation N1 Rb1_PPP1R26P1neo mice. Genomic DNA was digested with BamHI and hybridized with probe KIAA_5’ext, resulting in a 13 kb wildtype fragment and a 3.3 kb fragment of the targeted allele. C) Genotyping of generation N2 mice by PCR. To test for presence of PPP1R26P1 and wildtype alleles, a three-primer PCR was used (primers 1, 2 and 3, indicated by arrows in A), yielding a 210 bp product for the wildtype allele (primers 1 and 3) and a 362 bp product for the PPP1R26P1 allele (primers 2 and 3). A separate PCR was used for detection of the neomycin selection cassette, resulting in a 522 bp product for the cassette, if present. Black boxes: exons of the Rb1 gene, striped box: neomycin selection cassette, stippled grey box: PPP1R26P1, black triangles: loxP511 sites; black horizontal line: Southern blot probe; arrows: PCR primer, indicating direction; restriction sites: St: StuI, B: BamHI, X: XhoI.