Figure 1.
Generation of Trip12 mutant mice.
(A) Schematic representation of the Trip12 gene targeting construct. Exon 33 of Trip12 genomic DNA containing the Cys residue that is required for ubiquitination was replaced in frame with FLAG and HA epitope tag sequences. Thus, the Trip12 mutant protein has FLAG and HA epitope tags instead of ubiquitination activity. Solid rectangles represent the exons of the Trip12 gene. (B) Southern blot analysis of the targeted allele in heterozygote Trip12 mutant ES cells. (C) Western blot analysis of Trip12 mutant protein expression in ES cells. (D) Whole mount immunohistochemistry of wild-type and heterozygous Trip12 embryos using an anti-HA antibody.
Table 1.
Genotypes of the offspring from Trip12+/mt intercrosses.
Figure 2.
Analysis of wild-type (+/+) and Trip12 mutant (mt/mt) embryos.
(A) Different developmental stages (E8.5 to E10.5) of wild-type (+/+) and Trip12 mutant (mt/mt) embryos. (×magnification) (B) The somite number of Trip12+/+, Trip12+/mt, and Trip12mt/mt embryos. The somite number of each embryo at E9.5 was scored by stereo microscopy. **, p<0.001; N.S., not significant. (C) Hematoxylin and eosin staining of sections of wild-type (+/+) and Trip12 mutant (mt/mt) embryos at different developmental stages (E7.5 to E9.5). Placental tissues at E9.5 are enlarged in the right panel. Scale bars represent 1 mm. (D) The expression levels of negative cell cycle regulators in wild-type (+/+) and Trip12 mutant (mt/mt) embryos. Total RNA was isolated from embryos, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3. **, p<0.001; N.S., not significant.
Figure 3.
Generation and characterization of Trip12 mutant (mt/mt) ES cells.
(A) Schematic representation of the Trip12 gene targeting construct (Targeted allele II). Solid rectangles represent the exons of the Trip12 gene. (B) Southern blot analysis of the targeted allele in Trip12 mutant (mt/mt) ES cells. (C) Western blot analysis of Trip12 mutant protein expression in ES cells. (D) Expression levels of Nanog and Oct-4 in Trip12 mutant (mt/mt) ES cells with or without LIF. The cells were cultured without LIF and harvested at the indicated time points. Total RNA was isolated from ES cells, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3. (E) Embryoid body (EB) formation of wild-type (+/+) or Trip12 mutant (mt/mt) ES cells. The cells were cultured in suspension in ES cell medium without LIF in 10-mm dishes for 10 days. Scale bars: 100 µm. Total RNA was isolated from the cells, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3.
Figure 4.
Cell cycle defects in Trip12 mutant (mt/mt) ES cells.
(A) Proliferation of Trip12 mutant (mt/mt) ES cells. The growth rate of wild-type or Trip12 mutant (mt/mt) ES cells was measured by the MTT assay. (B) Cell cycle progression in Trip12 mutant (mt/mt) ES cells was analyzed by flow cytometry. (C) The expression levels of negative cell cycle regulators in wild-type (+/+) or Trip12 mutant (mt/mt) ES cells. Total RNA was isolated from the cells, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3. (D) Western blot analysis of proteins in the p53 pathway in wild-type (+/+) or Trip12 mutant (mt/mt) ES cells.
Figure 5.
Loss of the ubiquitin ligase activity of Trip12 induces BAF57 protein accumulation in ES cells.
(A) The BAF57 expression levels in wild-type (+/+) or Trip12 mutant (mt/mt) ES cells. The BAF57 protein levels were determined by immunoblotting, and the signal intensity was quantified. (B) Co-immunoprecipitation of Trip12 with BAF57. Trip12mt was immunoprecipitated from Trip12mt/mt ES cell lysate with an anti-FLAG antibody, and the immunoprecipitates were immunoblotted with the indicated antibodies. (C) Trip12 regulates the turnover of BAF57. The cells were treated with 100 µg/ml cycloheximide and lysed at various time points. The protein lysates were separated by SDS-PAGE and detected by immunoblotting. (D) Wild type or mutant Trip12 was ectopically expressed with or without myc-tagged BAF57. Myc-BAF57 was immunoprecipitated with anti-myc antibody and immunoblotted with anti-Trip12 or anti-myc antibody. (E) A comparison of the mouse whole-genome RNA expression profile between wild-type and Trip12 mutant (mt/mt) ES cells. DAVID and the annotation sources GOTERM_BP2 (Biological process) were used to identify the functional categories.