Fig. 1.
Genotyping and physical appearance of mice used in this study. (A) Isolated skin fibroblasts from 2 age-matched mice of p53−/− (p53-KO) and p53−/−Cry1−/−Cry2−/− (TRI-KO) genotypes were used for analysis. (Left) Genotyping by genomic PCR. For each gene, an anchor and a knockout or wild-type allele-specific primers were used. Genomic DNA from the tail of a triple heterozygous mouse was used as a positive control for each set of primer pairs. Lane 1, DNA from a triple heterozygous mouse; lanes 2 and 3, DNA from 2 p53-KO fibroblasts; lanes 4 and 5, DNA from 2 TRI-KO fibroblasts; lane 6, no genomic DNA. M, molecular size markers. (Right) Immunoblot of fibroblast lysates. 50 μg of protein from lysates of cultured fibroblasts were used for immunoblotting. Lanes 1 and 2, lysates from immortalized Cry1−/−Cry2−/− and wild-type (WT) fibroblasts, respectively (15); lanes 2 and 3, lysates from fibroblasts shown in Left lanes 2 and 3; lanes 5 and 6, lysates from fibroblasts shown in Left lanes 4 and 5. A protein band cross-reacting with Cry2 antibody (indicated by *) is shown as a loading control. (B) Physical appearance of mutant mice. TRI-KO (top) and p53-KO (bottom) at 8 weeks of age appear anatomically normal but the TRI-KO mutants are smaller and weigh ≈20% less than the p53-KO mice.