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note
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The targeting vector for the inactivation of the RORγ gene was constructed using the PL2-Neo vector by replacing the exon encoding the DNA-binding domain of RORγ with the neomycin resistance gene. Three of 150 G418-resistant E14 ES cell clones were positive for homologous recombination. Two of these were used to generate chimeric founder mice by microinjection into C57BL/ 6J blastocysts (10).
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0342931698
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Supplemental material is available at www. sciencemag.org/feature/data/1048806.shl
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6 cells) were washed twice with PBS and then resuspended in 300 μl of PBS. Cold ethanol (700 μl) was slowly added to the resuspended cells while vortexing. The cells were incubated on ice for 1 hour and washed twice with PBS. Fixed cells were resuspended in 1 ml of staining buffer (1 X PBS, 0.1% NP-40, 0.1 mg/ml RNase A, and 0.05 mg/ml PI) and incubated on ice for 20 min. DNA content of cells was analyzed using flow cytometry.
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13
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0343367055
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note
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32P]ATP at 30°C for 30 min. The reaction was terminated by heat inactivation at 100°C for 5 min. The reaction mixture was resolved on a 12% SDS-PAGE gel which was then dried and autoradiographed for 4 hours.
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note
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For visualization of lymph nodes, 250 μl of 1% Evans blue dye (Sigma) was injected into the hind footpads of mice. After 30 min, mice were sacrificed and superficial inguinal lymph nodes were visualized and documented under a dissecting microscope using a digital camera. For mesenteric lymph node visualization, mice were sacrificed 1 to 1.5 hours after injection of Evans blue dye. For analysis of precursors, the mesentery of each embryo was dissected away from the intestines and both were digested separately using collagenase type IV (Sigma) [0.5 mg/ml in PBS containing 2% fetal bovine serum (FBS)] for 30 min. The cells were washed and subsequently stained with CD45-FITC, CD4-PE, and CD3-biotin (Pharmingen), washed, and incubated with Streptavidin-CyChrome (Pharmingen). After washing, the cells were resuspended in PBS containing 2% FBS and 7-AAD, to discriminate live versus dead cells.
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0342931694
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note
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E12.5 mouse embryos were dissected and fixed in 4% paraformaldehyde, 0.1 M phosphate buffer (PB) (pH 7.4) at 4°C for 1 to 2 hours, washed with PBS, equilibrated with 30% sucrose, 0.1 M PB, and mounted in OCT embedding medium. Frozen 11 μM cryostat sections were mounted on SuperFrost Plus slides (Fisher) and subsequently analyzed by in situ hybridization or immunohistochemistry (10). Antibodies used were hamster monoclonal anti-RORγ (diluted 1:10) and Alexa-488-conjugated anti-CD4 (diluted 1:200). Cy3-conjugated goat anti-hamster (Jackson Immuno Research Laboratory) was used at 1:700 dilution. Images were collected on a Zeiss Axioplan 2 microscope using Slidebook software (Innovative Imaging). A digoxigenin-labeled antisense riboprobe complementary to the RORγ cDNA was synthesized according to supplier protocol (Boehringer Mannheim). The hamster monoclonal antibody against murine RORγ was prepared using a His-tagged RORγ expressed in bacteria as immunogen (10).
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0342497516
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note
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We thank F. Weis-Garcia for preparation of mAbs, S. Jung and R. Lang for help with microscopy, S. Smith for the TUNEL analysis, J. McMicking for assistance with intestinal lymphoid organ analysis, S. Korsmeyer for Bcl-xL transgenic mice, P. Rennert and H. Petrie for helpful discussions, and Y. Choi, W. Ellmeier, and L. Johnston for comments on the manuscript. Supported by an NIH Molecular Oncology and Immunology training grant (Z.S.), the Royal Netherlands Academy of Arts and Sciences (R.E.M.), a postdoctoral fellowship from the Irvington Institute (Y.-R.Z.), and the Howard Hughes Medical Institute (D.R.L.).
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