(A) A diagram of Mdm2 protein and approximate binding domains for p53, ARF, and several ribosomal proteins.
(B) Schematic representation of Mdm2C305F targeting strategy. Exons 7-12 of Mdm2 are shown in black boxes. The targeting vector contains negative selection marker thymidine kinase (TK) and positive selection marker neomycin resistant gene (neo). Two loxP sites are shown as triangles.
(C) Southern blot analysis was used to screen ES clones for the targeted allele. BamH1 digestion generates a 9.5 kb fragment from the wild type allele. The targeted allele gives rise to two BamH1 fragments of 4.8 kb and 6.5 kb, respectively.
(D) DNA from ES cells positive for the recombined allele identified was analyzed for the presence of Mdm2C305F mutation by PCR amplification and Spe1 digestion.
(E) Shown are expected and observed birth ratios from a total of 151 mice obtained from Mdm2+/m intercross.
(F) Kaplan-Mierer survival curve for Mdm2+/+ (+/+), Mdm2+/C305F (+/m) and Mdm2C305F/C305F (m/m) mice are shown.