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. 2005 Jul 29;102(32):11296–11301. doi: 10.1073/pnas.0505053102

Fig. 2.

Fig. 2.

Generation and characterization of Mad2–/– p53–/– MEF cell lines. (A) Numbers of E10.5 embryos with the indicated genotypes. Embryos from natural matings were recovered at E10.5. (B) Genotypes of MEF cell lines. (C) MEF DNA was subjected to PCR at the Mad2 (lanes 1–4) and p53 loci (lanes 5 and 6). Mad2+/+ p53–/– (AB103), Mad2+/– p53–/– (AB100) and Mad2–/– p53–/– (AB98) cell lines were derived from embryos of the same litter. The bands for mutant and wild-type alleles are indicated by “m” and “w,” respectively. Y-sacAB152, genotyping of the yolk sac of AB152. (D) MEFs were grown in culture and imaged to compare cell morphology and growth. Shown are phase-contrast images (Upper) and, at higher magnification, phase-contrast/fluorescence images (Lower) taken of cells expressing H2B–GFP to visualize chromatin. Images were taken with a ×10, 0.4 numerical aperture objective. (Scale bars: Upper, 100 μm; Lower, 10 μm.) (E) Mad2 mRNA levels as assayed by RT-PCR. Total RNA from AB103, AB100, and AB98 MEF lines was isolated, transcribed into cDNA, and subjected to PCR by using Mad2-specific (lanes 1–3) and GAPDH-specific (lanes 3–5) oligonucleotides. (F) Mad2 and γ-tubulin levels in lysates of 3T3 cells, AB103, AB100, and AB98 MEF lines were determined by using Western blotting; 2.5 ng of recombinant mouse Mad2 protein (lane 1) was loaded as a positive control. The Mad2 polyclonal antibody recognizes an uncharacterized band (*) in 3T3 cells but not in other MEFs that electrophoreses more slowly than the Mad2 band. (G) Checkpoint status as monitored by live cell imaging. AB103 and AB98 expressing H2B–GFP were treated with 330 nM nocodazole and imaged by time-lapse microscopy. The graph shows the percentage of cells that have undergone anaphase B and exited mitosis 60 min after NBD.