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Development and characterization of immortalized ovine endometrial cell lines.

Center for Animal Biotechnology and Genomics, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, and Department of Animal Science, Texas A&M University, College Station, Texas 77843, USA.
Biology of Reproduction (Impact Factor: 3.45). 12/1999; 61(5):1324-30.
Source: PubMed

ABSTRACT The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.6-0.8 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for more than 40 passages. Phase-contrast microscopy revealed that LE and GE cells exhibited a cobblestone morphology whereas immortalized ST cells were spindle shaped. The epithelial origin of LE and GE was confirmed by positive cytokeratin immunostaining, and ST cells were vimentin positive. All cell lines were negative for smooth muscle alpha-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFNtau) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFNtau. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium.

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    • "We used the ovine immortalized endometrial luminal epithelial (LE) cells and stromal (STR) cells to determine IFNT signaling. These LE and STR cells expressed IFNT receptors IFNR1 and IFNR2 and responded to IFNT treatment (Chen et al., 2007; Johnson et al., 1999; Stewart et al., 2001). The LE and STR cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/ F12) medium containing 5% dextran charcoal treated fetal bovine serum (DC-FBS) and 100 U penicillin/ml, 100 lg streptomycin/ml and 2.5 lg amphotericin B/ml in humidified 5% CO 2 and 95% air at 37 °C. "
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    • "To validate our results from the multiple microarray analyses, we performed real-time PCR experiments on five selected genes. ISG15 was chosen as a positive control for validating the experimental procedure because it has been previously shown to be up-regulated by IFNT in ovine LE cells (Johnson et al. 1999c). We selected PTGS2, IGF2, and hypoxia-inducible factor-1a subunit (HIF1A), because we had previously demonstrated these genes to be down-regulated in endometrium from ewes that were either pregnant or, alternatively, treated with IFNT (Chen et al. 2006). "
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    • "Immortalized ovine uterine endometrial LE cells were cultured as described previously (Johnson et al. 1999c). Bovine endometrial (BEND) cells (Johnson et al. 1999a) were kindly provided by Dr Thomas R Hansen (Colorado State University, Fort Collins, CO, USA). "
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