Distribution of <i>Cul4b</i> genotypes in progeny of <i>Cul4b</i>^+/flox/<i>Cul4b</i>^+/null;<i>EIIa-Cre</i>^+/− females.

<p>Litters were dissected at the times shown and genotyped by PCR as described in Materials and Methods.</p>a<p>ND indicates that the <i>Cul4b</i> genotype could not be determined by PCR.</p

Growth retardation of <i>Cul4b</i> heterozygous mice during embryonic development.

<p>(A) Bodyweights of <i>Cul4b</i> heterozygous mice and littermate wild-type females after birth. Data were presented as mean±SD. N = 8, *: p<0.05; **: p<0.01; ***: p<0.001. (B–E) Representative photographs of <i>Cul4b</i> heterozygous embryos and littermate wild-type controls at 9.5 (B), 10.5 (C), 12.5 (D) and 14.5 (E) dpc. The bar represents 1 mm in (B–C) and 2 mm in (D) and (E), respectively.</p

Characterization of X chromosome inactivation by Cul4b expression in heterozygous mice.

<p>(A–C) Percentages of cells positive for Cul4b of <i>Cul4b</i> heterozygous mice and littermate wild-type female controls at 4 months (A), 3 weeks (B) and newborn (C). More than 2,000 cells of each tissue were scored. Hi, hippocampus; Ki, kidney; Li, liver; Lu, lung. Data were presented as mean±SD. *: p<0.05; **: p<0.01; ***: p<0.001. (D–E) Representative images of liver (D) and hippocampus (E) at 3 weeks stained with an antibody against Cul4b. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels. (F) Immunohistochemistry of paraffin sections of <i>Cul4b</i> heterozygous embryos at 7.5 dpc with an anti-Cul4b antibody. Embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. Middle and right panels are the higher magnification of the left panel.</p

Morphology and histology of placentas of wild-type, <i>Cul4b</i> heterozygous and absorbed embryos at 14.5 dpc.

<p>(A) Representative photographs of placentas of wild-type, <i>Cul4b</i> heterozygous and absorbed embryos at 14.5 dpc. (B) H&E staining of radial sections of placentas. sp, spongiotrophoblast layer; la, labyrinthine layer. Lower panels are the higher magnification of the upper panels. (C) Immunohistochemisty of radial sections of placentas with an antibody to PECAM, an angiogenesis marker. Middle panels are the higher magnification of the upper panels, and lower panels are the higher magnification of the middle panels.</p

Decreased proliferation and increased apoptosis in <i>Cul4b</i> null embryos.

<p>(A) Paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc were stained with an antibody against Ki67, a proliferation marker. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels. (B) Paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc were analyzed by immunostaining against BrdU, and counterstained with DAPI. Lower panels are the higher magnification of the upper panels. (C) Paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc were analysed by TUNEL assay for labeling apoptotic cells, and counterstained with DAPI. Lower panels are the higher magnification of the upper panels. (D) Paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc were stained with an antibody against cyclin E. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels.</p

Morphology and histology of <i>Cul4b</i> null embryos.

<p>(A) Uterus excised from pregnant female at 12.5 dpc. Arrows indicate absorbed embryos. (B) Photomicrographs of E7.5 embryos dissected from surrounding deciduas tissue of the same uterus. The two embryos on the left appear normal in size and morphology and the two on the right were much smaller and were partially deteriorated. The bar represents 200 µm. (C) H&E staining of paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc. Littermate embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. The genotype of each embryo was determined by immunohistochemistry using an anti-Cul4b antibody, as shown below. (D) Immunohistochemistry of paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc with an anti-Cul4b antibody. (E–F) Photomicrographs with higher magnification of the stained section shown in (D).</p

Generation of <i>Cul4b</i> flox mice.

<p>(A) Strategy for generation of <i>Cul4b</i> floxed targeting vector. On the top was shown the wild-type allele of <i>Cul4b</i> gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. <i>BamHI</i> (labeled B) and <i>XbaI</i> (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. <i>BamHI</i> digested DNA was hybridized with 5′ probe and <i>XbaI</i> digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of <i>Cul4b</i> gene from the brain tissue of brain-specific knockout mice (<i>Cul4b</i>^flox/Y;<i>Nesin-Cre</i>). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of <i>Cul4b</i> mRNA isolated from brain tissues of wild-type males (<i>Cul4b</i>^+/Y;<i>Nestin-Cre</i>^+/−), wild-type females (<i>Cul4b^+/+</i>;<i>Nestin-Cre</i>^+/−), heterozygous females (<i>Cul4b</i>^+/flox;<i>Nestin-Cre</i>^+/−), and conditional knock-out males (<i>Cul4b</i>^flox/Y;<i>Nestin-Cre</i>^+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.</p