Although smaller bands of indeterminate origin are detected in wild-type and GT lysates. Right panel: an anti-USO1 antibody whose epitope is carboxyl-terminal (Cterm.) to the site of the USO1-Beta-Geo fusion detects full length USO1 protein (arrow) in all lysates. doi:10.1371/journal.pone.0050530.gUSO1 Inactivation in the MouseFigure 3. Fetal death occurs by E8.5 in embryos that are homozygous for the Uso1 GT alleles. A) Table indicating the frequencies of genotypes in fetuses/blastocysts recovered from heterozygous Uso1 GT mating pairs. Anticipated genotypes included WT (+/+), heterozygous GT (+/ GT), and 1531364 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) Genotypes of E3.5 blastocysts obtained from a heterozygous AW0562 GT mating pair. One DprE1-IN-2 web blastocyst was homozygous for the GT (lane 1), while 4 others were heterozygous. C) 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. D) 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 carboxylterminal 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 Uso1 GT allele (GT/GT). doi:10.1371/journal.pone.0050530.gX-gal staining to identify Beta-galactosidase activityPrimary skin fibroblasts and HEK293T (Human embryonic kidney cells, ATCC CRL1573) cells were plated onto 8-chamber culture slides (BD Biosciences). Upon reaching confluence, cells were washed with PBS and fixed in ice cold X-Gal fixative (PBS containing 0.2 glutaraldehyde, 5 mM EDTA and 2 mM MgCl2) for 10 minutes. Subsequently cells were washed 3x for 5 minutes with 0.5 ml wash solution (PBS containing 2 mM MgCl2 and 0.02 NP-40). X-gal staining was performed overnight in the dark (X-gal staining solution: PBS containing 5 mM 1662274 Potassium-ferrocyanide, 5 mM Potassium-ferri-cyanide, 2 mM MgCl2, 0.02 NP-40 and 2 mg/ml X-Gal). Cells were subsequently washed 3x with PBS and kept in PBS at 4uC. As a positive control for Betagalactosidase activity the HEK293T cells were transfected with 0.5 mg of pSV40-LacZ (Promega).subsequently separated on a NUPAGE 3? Tris-Acetate gel (Invitrogen) and transferred overnight at 15 V onto a PVDF membrane (Invitrogen). Immunodetection of USO1 was performed using the Western breeze system (Invitrogen). An amino terminal anti-USO1 antibody (NB100-74483; Novus Biologicals) and a carboxyl-terminal USO1 antibody (13509-1-AP; Proteintech) were each used at a 1/1000 dilution.Retrieval of blastocysts from GT breeding TA02 site pairsHeterozygous GT breeding pairs were checked daily for mating by identification of vaginal plugs. When a vaginal plug was observed, the female was euthanized 72 hrs later, the uterus was removed and placed in a 60 mm dish containing 1 ml of M2 medium (Sigma), and t.Although smaller bands of indeterminate origin are detected in wild-type and GT lysates. Right panel: an anti-USO1 antibody whose epitope is carboxyl-terminal (Cterm.) to the site of the USO1-Beta-Geo fusion detects full length USO1 protein (arrow) in all lysates. doi:10.1371/journal.pone.0050530.gUSO1 Inactivation in the MouseFigure 3. Fetal death occurs by E8.5 in embryos that are homozygous for the Uso1 GT alleles. A) Table indicating the frequencies of genotypes in fetuses/blastocysts recovered from heterozygous Uso1 GT mating pairs. Anticipated genotypes included WT (+/+), heterozygous GT (+/ GT), and 1531364 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) 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. C) 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. D) 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 carboxylterminal 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 Uso1 GT allele (GT/GT). doi:10.1371/journal.pone.0050530.gX-gal staining to identify Beta-galactosidase activityPrimary skin fibroblasts and HEK293T (Human embryonic kidney cells, ATCC CRL1573) cells were plated onto 8-chamber culture slides (BD Biosciences). Upon reaching confluence, cells were washed with PBS and fixed in ice cold X-Gal fixative (PBS containing 0.2 glutaraldehyde, 5 mM EDTA and 2 mM MgCl2) for 10 minutes. Subsequently cells were washed 3x for 5 minutes with 0.5 ml wash solution (PBS containing 2 mM MgCl2 and 0.02 NP-40). X-gal staining was performed overnight in the dark (X-gal staining solution: PBS containing 5 mM 1662274 Potassium-ferrocyanide, 5 mM Potassium-ferri-cyanide, 2 mM MgCl2, 0.02 NP-40 and 2 mg/ml X-Gal). Cells were subsequently washed 3x with PBS and kept in PBS at 4uC. As a positive control for Betagalactosidase activity the HEK293T cells were transfected with 0.5 mg of pSV40-LacZ (Promega).subsequently separated on a NUPAGE 3? Tris-Acetate gel (Invitrogen) and transferred overnight at 15 V onto a PVDF membrane (Invitrogen). Immunodetection of USO1 was performed using the Western breeze system (Invitrogen). An amino terminal anti-USO1 antibody (NB100-74483; Novus Biologicals) and a carboxyl-terminal USO1 antibody (13509-1-AP; Proteintech) were each used at a 1/1000 dilution.Retrieval of blastocysts from GT breeding pairsHeterozygous GT breeding pairs were checked daily for mating by identification of vaginal plugs. When a vaginal plug was observed, the female was euthanized 72 hrs later, the uterus was removed and placed in a 60 mm dish containing 1 ml of M2 medium (Sigma), and t.
