Generation of INKBRITE mouse model

Three H2B-GFP cassettes were cloned in tandem into one cassette with each separated by a 2A self-cleaving peptide (3X 2A-H2B-GFP). The 3X-2A-H2B-GFP fusion cassette was then inserted into bacterial artificial chromosome (BAC) containing the Cdkn2a locus (BAC clone RP24-322D20 from the RPCI-24 C57BL/6J male mus musculus BAC library http://bacpac.chori.org/library.php). The 3X 2A-H2B-GFP cassette was inserted in frame with the p16INK4A reading frame in exon 2 at the identical insertion site as the 3MR model as previously described (16). The insertion site prevents the production of a full length p16INK4a transcripts and also produces a premature stop codon in the p19ARF reading frame within the shared exon 2 of the two transcripts, so no mature transcripts of p16 or p19 are produced by the BAC. Furthermore, the insertion frame of the cassette would produce a frame shift mutation in the p19ARF reading frame without translation of the H2B-GFP protein, thus only p16INK4A expression is reported. A neo cassette 3’ to the 3X 2A-H2B-GFP cassette used for selection was removed before the BAC injection.