He uterine horns were flushed using a 20 gauge needle with 0.5 ml of pre-warmed (37uC) M2 medium 23388095 to obtain blastocysts. Blastocysts were identified microscopically, retrieved with a 0.8?.106100 mm capillary tube (Kimax), and placed individually into different gelatin-coated chambers filled with 0.2 ml of blastocyst medium (DMEM/15 FBS/nonessential amino acids; Invitrogen). Eight-chamber Tubastatin A web culture slides (BD Biosciences), pre-coated with 0.1 gelatin (Sigma) for 30 minutes at room temperature, were used. DNA was extracted from individual blastocysts after 3 days of culture (Arcturus PicoPure DNA extraction kit, Applied Biosystems) and used for WT and GT allele genotyping.Immuno-detection of USO1 in cell lysatePrimary skin fibroblasts were lysed in RIPA buffer (Sigma) containing 1x EDTA free protease inhibitor cocktail (Thermoscientific) for 10 minutes on ice. One ml of lysis buffer was used to lyse fibroblasts collected from a confluent 75 cm2 culture flask. Lysates were then cleared of debris by centrifugation (16,1006g, 2 min). The protein concentration in each lysate was measured using the Bradford assay (Quick Start Bradford Dye reagent, Biorad) and RIPA buffer was then added to equalize the protein 16985061 concentration across all lysates. Equal amounts of lysates wereUSO1 Inactivation in the MouseFigure 4. Blastocysts that are Madrasin web homozygous for a Uso1 GT allele have a dispersed Golgi architecture. Confocal laser scanning double immunofluorescence images (magnification 400x) of cells within cultured E3.5 blastocysts that were recovered from heterozygous Uso1 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. doi:10.1371/journal.pone.0050530.gImmuno-detection of USO1 and GM-130 in cultured blastocystsAfter 3 days in culture, blastocysts were washed with 0.5 ml PBS and fixed to the glass slide with 0.5 ml of 4 paraformaldehyde for 20 minutes at room temperature. Cells were subsequently washed twice with PBS, twice with 0.1M NH4Cl and twice with PBS. Primary antibody incubation was performed overnight at 4uC in PBS containing 5 FBS, 2 BSA and 0.1 Saponin. Cells were washed 3x with 0.5 ml PBS and incubated with secondary antibody in PBS for 30 minutes at room temperature. Cells were subsequently washed 3x with 0.5 ml PBS and mounted in DAPI Fluoromount G (Southern Biotech). Primary antibodies were used in a 1/1,000 dilution and secondary antibodies were used in a 1/10,000 dilution. Primary antibodies used were mouse anti-GM130 (610822, BD Transduction laboratories) and rabbit anti-USO1 (13509-1-AP, Proteintech). Secondary antibodies used were Cy3 anti-rabbit IgG (XG6157cy3, ProScience) and Fluorescein anti-mouse IgG (XR9760, ProScience). Fluorescence images were obtained using a NikonRi1 camera mounted to a Nikon Eclipse 80i microscope. Confocal laser scanning microscopy was performed using the Zeiss LSM 780 system. Mutant and control pictures were equally adjusted for brightness and contrast using Adobe Photoshop CS3.Results Mice heterozygous for the AW0562 or YTA025 GT.He uterine horns were flushed using a 20 gauge needle with 0.5 ml of pre-warmed (37uC) M2 medium 23388095 to obtain blastocysts. Blastocysts were identified microscopically, retrieved with a 0.8?.106100 mm capillary tube (Kimax), and placed individually into different gelatin-coated chambers filled with 0.2 ml of blastocyst medium (DMEM/15 FBS/nonessential amino acids; Invitrogen). Eight-chamber culture slides (BD Biosciences), pre-coated with 0.1 gelatin (Sigma) for 30 minutes at room temperature, were used. DNA was extracted from individual blastocysts after 3 days of culture (Arcturus PicoPure DNA extraction kit, Applied Biosystems) and used for WT and GT allele genotyping.Immuno-detection of USO1 in cell lysatePrimary skin fibroblasts were lysed in RIPA buffer (Sigma) containing 1x EDTA free protease inhibitor cocktail (Thermoscientific) for 10 minutes on ice. One ml of lysis buffer was used to lyse fibroblasts collected from a confluent 75 cm2 culture flask. Lysates were then cleared of debris by centrifugation (16,1006g, 2 min). The protein concentration in each lysate was measured using the Bradford assay (Quick Start Bradford Dye reagent, Biorad) and RIPA buffer was then added to equalize the protein 16985061 concentration across all lysates. Equal amounts of lysates wereUSO1 Inactivation in the MouseFigure 4. Blastocysts that are homozygous for a Uso1 GT allele have a dispersed Golgi architecture. Confocal laser scanning double immunofluorescence images (magnification 400x) of cells within cultured E3.5 blastocysts that were recovered from heterozygous Uso1 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. doi:10.1371/journal.pone.0050530.gImmuno-detection of USO1 and GM-130 in cultured blastocystsAfter 3 days in culture, blastocysts were washed with 0.5 ml PBS and fixed to the glass slide with 0.5 ml of 4 paraformaldehyde for 20 minutes at room temperature. Cells were subsequently washed twice with PBS, twice with 0.1M NH4Cl and twice with PBS. Primary antibody incubation was performed overnight at 4uC in PBS containing 5 FBS, 2 BSA and 0.1 Saponin. Cells were washed 3x with 0.5 ml PBS and incubated with secondary antibody in PBS for 30 minutes at room temperature. Cells were subsequently washed 3x with 0.5 ml PBS and mounted in DAPI Fluoromount G (Southern Biotech). Primary antibodies were used in a 1/1,000 dilution and secondary antibodies were used in a 1/10,000 dilution. Primary antibodies used were mouse anti-GM130 (610822, BD Transduction laboratories) and rabbit anti-USO1 (13509-1-AP, Proteintech). Secondary antibodies used were Cy3 anti-rabbit IgG (XG6157cy3, ProScience) and Fluorescein anti-mouse IgG (XR9760, ProScience). Fluorescence images were obtained using a NikonRi1 camera mounted to a Nikon Eclipse 80i microscope. Confocal laser scanning microscopy was performed using the Zeiss LSM 780 system. Mutant and control pictures were equally adjusted for brightness and contrast using Adobe Photoshop CS3.Results Mice heterozygous for the AW0562 or YTA025 GT.