Generation of mice that transmit GT alleles at the <i>Uso1</i> locus.

<p><b>A</b>) Schematic representation of the gene trap insertions into <i>Uso1</i>. The <i>Uso1</i> exons upstream and downstream of the GT are shaded grey. These are exons 10 and 11 for AW0562 and exons 12 and 13 for YTA025. The GT contains intron 1 and a strong splice acceptor (SA) from the engrailed locus (En2-intron1-SA), coding sequence for Beta-Geo, and a poly-A (pA) addition site. Sites that can be used for Cre-mediated (Lox71 and LoxP) and Flpe-mediated recombination (Frt) are also contained within the GT. Primer pairs used for RT-PCR and genotyping are numbered and color-coded and their approximate locations within the exon, intron, or GT vector are indicated. <b>B</b>) Genotyping of agouti offspring from chimeric males generated using AW0562 and YTA025 ES cells. Pups carrying the GT allele were identified by PCR amplification of a fragment of the Beta-Geo cassette (primer pair 1 – 2, green). <b>C</b>) RT-PCR confirming splicing of the GT to the <i>Uso1</i> gene in both cell lines (black primer pairs). RNA was extracted from primary skin fibroblasts established from wild-type (WT) and GT heterozygous (HET) mice. Top left panel: AW0562 wild-type (WT) allele, primer pair 3 – 5. Bottom left panel: AW0562 GT allele, primer pair 3 – 4. Top right panel: YTA025 WT allele, primer pair 3 – 5. Bottom right panel: YTA025 GT allele, primer pair 3 – 4. <b>D</b>) Genotyping of offspring from matings between wild type mice and AW0562 or YTA025 GT heterozygous mice confirming the insertion of the GT in the introns immediately following the trapped <i>Uso1</i> exons. Top left panel: AW0562 WT allele, primer pair 6 – 8 (red). Bottom left panel: AW0562 GT allele, primer pair 6 – 7 (red). Top right panel: YTA025 WT allele, primer pair 9 – 11 (orange). Bottom right panel: YTA025 GT allele, primer pair 9 – 10 (orange).</p

Blastocysts that are homozygous for a <i>Uso1</i> GT allele have a dispersed Golgi architecture.

<p>Confocal laser scanning double immunofluorescence images (magnification 400x) of cells within cultured E3.5 blastocysts that were recovered from heterozygous <i>Uso1</i> GT mating pairs. Antibodies recognizing epitopes in the USO1 carboxyl-terminal domain (red fluorescence) or the Golgi protein GM130 (green fluorescence) were used. DAPI staining was used to mark cell nuclei (blue fluorescence). In cells from blastocysts containing immuno-detectable USO1, GM130 localizes near the cell nuclei, overlapping with USO1 localization. In contrast, in cells from blastocysts that lack immuno-detectable USO1 protein, GM130 does not localize near the nucleus but is more dispersed throughout the cytoplasm.</p

Fetal death occurs by E8.5 in embryos that are homozygous for the <i>Uso1</i> GT alleles.

<p><b>A</b>) Table indicating the frequencies of genotypes in fetuses/blastocysts recovered from heterozygous <i>Uso1</i> GT mating pairs. Anticipated genotypes included WT (+/+), heterozygous GT (+/GT), and homozygous GT (GT/GT). No homozygous GT fetuses were observed at E11.5, E9.5, and E8.5. In contrast, 2 out of 11 E3.5 blastocysts were homozygous for the GT. <b>B</b>) Genotypes of E3.5 blastocysts obtained from a heterozygous AW0562 GT mating pair. One blastocyst was homozygous for the GT (lane 1), while 4 others were heterozygous. <b>C</b>) Table indicating the frequencies of immuno-detectable USO1 protein in cultured E3.5 blastocysts from wild-type x heterozygous YTA025 GT and heterozygous YTA025 GT x heterozygous YTA025 GT mating pairs. Immuno-detection was performed using an antibody that recognizes an epitope in the USO1 carboxyl-terminal domain. <b>D</b>) Photomicrograhs of double immunofluorescence images of cultured E3.5 blastocysts recovered from a heterozygous YTA025 GT mating pair. Antibodies that recognize epitopes in the USO1 carboxyl-terminal domain (red fluorescence) or the Golgi protein GM130 (green fluorescence) were employed. DAPI staining was used to mark cell nuclei (blue fluorescence). The upper panels depict fluorescence patterns that represent a blastocyst that is either wild-type (+/+) or heterozygous for the GT allele (+/GT). The lower panels depict fluorescence patterns that represent a blastocyst that is homozygous for the <i>Uso1</i> GT allele (GT/GT).</p

μCT measurements of cancellous and cortical bone properties in the femur of ground control (GC) and tail suspended (TS) mice.

<p>(A) Bone volume fraction, (B) trabecular number, and (C) trabecular thickness were measured in the distal femur metaphysis. (D) Cortical area, (E) medullary area, and (F) total tissue area were measured in the mid-diaphyseal femur. (F) Representative μCT reconstructions of the distal femur from GC ad TS mice within each genotype. The anterior and posterior thirds of each reconstruction have been removed digitally to reveal the metaphyseal spongiosa. *p<0.05 for tail suspended mice vs genotype-matched control mice. <i>n</i> = 8/group.</p

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μCT measurements of cancellous and cortical bone properties in the femur from mice that underwent ovariectomy surgery (Ov) or sham surgery (Sh).

<p>(A) Bone volume fraction, (B) trabecular number, and (C) trabecular thickness were measured in the distal femur metaphysis. (D) Cortical area, (E) medullary area, and (F) total tissue area were measured in the mid-diaphyseal femur. (F) Representative μCT reconstructions of the distal femur from OVX- and sham-operated mice within each genotype. The anterior and posterior thirds of each reconstruction have been removed digitally to reveal the metaphyseal spongiosa. *p<0.05 for OVX vs. sham within genotype. <i>n</i> = 8/group, except for G171V mice (<i>n</i> = 7).</p

μCT measurements of cancellous and cortical bone properties in the femur from mice that underwent intramuscular injection of saline (SL) into the left lower limb and botox (BX) into the right lower limb.

<p>(A) Bone volume fraction, (B) trabecular number, and (C) trabecular thickness were measured in the distal femur metaphysis. (D) Cortical area, (E) medullary area, and (F) total tissue area were measured in the mid-diaphyseal femur. (F) Representative μCT reconstructions of the distal femur from SL- ad BX-treated limbs within each genotype. The anterior and posterior thirds of each reconstruction have been removed digitally to reveal the metaphyseal spongiosa. *p<0.05 for botox-treated limb vs. saline-treated limb within genotype. <i>n</i> = 8/group.</p

Endogenous <i>Wisp3</i> expression and <i>Wisp3</i>^{<i>GFP-Cre</i>} expression are not identical.

<p>(Upper panel) RT-PCR amplimers indicating the presence of <i>Wisp3</i> transcript in total RNA from a several different mouse tissues. A schematic of <i>Wisp3</i> mRNA is indicated (not drawn to scale) along with the locations of the intron-spanning PCR primers and the expected amplimer size for correctly splice mRNA (Lower panel). A schematic of the <i>ROSA26</i>^mTmG allele before and after Cre-recombination (not drawn to scale) along with the locations of the PCR primers and the expected amplimer sizes for the non-recombined and recombined alleles. PCR amplimers indicating non-recombined <i>ROSA26</i>^mTmG DNA (upper gel) in all tissues and Cre-mediated recombination (lower gel arrowheads) in testis, heart, and brain recovered from <i>Wisp3</i>^{+/GFP- Cre};<i>ROSA26</i>^+/mTmG mice (floxed and recombined template DNA serves as controls for the two primer pairs). Note that there is poor correlation between endogenous <i>Wisp3</i> expression (upper panel) and <i>Wisp3</i>^GFP-Cre activity (lower panel).</p

<i>Wisp3</i>^GFP-Cre activity occurs in spermatocytes early during meiosis I.

<p>Fluorescence microscope images of seminiferous tubules from <i>Wisp3</i>^+/GFP-Cre;<i>ROSA26</i>^+/mTmG male mice at 5, 10, 14, 15, 17, 19 and 27 days-of-age (P5 – P27). Cell nuclei are imaged with DAPI dye. Spermatocytes expressing membrane bound Green Fluorescent Protein (GFP) instead of Tomato Fluorescent Protein (TFP), which indicates that Cre-mediated recombination has occurred, become visible by P15 and increase in abundance with age. Germ cells and spermatogonial cells, which reside near the periphery of seminiferous tubules, do not express GFP. The location of the GFP expressing cells in the seminiferous tubules, coupled with PCR evidence of recombination by P10 (data not shown), suggests that the <i>Wisp3</i>^GFP-Cre allele is expressed in spermatocytes between the leptotene and pachytene stages of male meiosis I.</p

Lineage tracing of SHP2-depleted chondrocytes in mouse vertebral growth plates following mosaic postnatal <i>Ptpn11</i> inactivation.

<p>Tissue sections of lumbar vertebral growth plates (GP) and intervertebral disks (IVD) from <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl</i>^/<i>fl</i> (Col2a1-cKO) or <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl/+</i> (Ctrl) mice that had been administered tamoxifen for 5 days, starting at w1, and sacrificed at w7. A-C: Tissue sections stained with Alcian Blue and Nuclear Fast Red. In Col2a1-cKO mice, the vertebral growth plates are enlarged and disorganized, with ectopic areas of ossification inside the growth plate (red arrow in B) and enchondroma-like lesions below the growth plate (C). D-G: Merged fluorescent images showing cells in which the <i>mTmG</i> reporter allele has (green fluorescence) or has not (red fluorescence) been recombined by Cre recombinase. Note that approximately 50% of growth plate chondrocytes fluoresce green, and in Col2a1-cKO mice, the expanded regions of the growth plate (E), lateral outgrowths (F) and enchondroma-like lesions (G) contain both green and red fluorescing cells. Dashed-white lines outline the cartilage. Scale bars  =  100 µm.</p