CK-35 ES cells were electroporated with the targeting construct linearized with Not I. Recombinant clones were identified by long-range PCR, confirmed by Southern blot, PCR, and DNA sequencing (primer sequences in table S4). Two independent recombinant clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping their offspring. Reverse transcription PCR (RT-PCR) of RNAs from Mdm4TM/TM MEFs showed that the mutant complementary DNA (cDNA) differed from an Mdm4 WT sequence only by the engineered missense mutation. The genotyping of p53+/−, p53+/31, and G3 Terc−/− mice was performed as previously described (5, 12). All experiments were performed according to Institutional Animal Care and Use Committee regulations.

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