PMC:13916 / 3448-5671
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/13916","sourcedb":"PMC","sourceid":"13916","source_url":"http://www.ncbi.nlm.nih.gov/pmc/13916","text":"Materials and methods:\nThe estrogen receptor-positive, BG-1 cell line [13], which contains an abundant amount of estrogen receptors (600 fmoles/100 μg DNA), was infected using a pLXSN retroviral vector (provided by AD Miller) containing an inverted partial human cDNA 900-base-pair sequence of BRCA1 (from nucleotide 121 in exon 1 to nucleotide 1025 in exon 11, accession #U14680). After 2 weeks of selection in 800 μg/ml of geneticin-G418 (Gibco/Life Technologies, Gaithersburg, MD, USA), BG-1 G418-resistant colonies were pooled, or individually isolated, and assayed for growth in the presence or absence of supplemented estrogen. Virally infected pooled populations of BG-1 cells were examined for BRCA1 message levels by ribonuclease protection assay (Fig. 1a). BRCA1 ribonuclease protection probe was made using an in vitro transcription kit (Ambion, Inc, Austin, TX, USA) as previously described [10] and derived clones were tested for protein levels by Western blot analysis using an anti-BRCA1 (Oncogene Research, Ab-1, Cambridge, MA, USA) antibody. Growth curve analysis of Infected populations and were pretreated for 5 days in phenol red-free, Dulbecco's modified eagle medium (DMEM)/F-12 medium (Gibco/Life Technologies) supplemented with 10% charcoal/dextran treated serum (Hyclone, Logan, UT, USA), then plated at 2.5 × 106 cells per 100mm dish in triplicate in the absence or presence of estrogen (10-8 mol/l; 17β-Estradiol; 1,3,5 (10) - Estratriene 3,17β-diol; Sigma, St Louis, MO, USA). For soft agar assay, clones were plated into 10 60-mm dishes at 1 × 105 cells/dish containing 0.3% bactopeptone agar with or without added estrogen (10-8 mol/l) in phenol red-free medium with 10% stripped serum in order to test for anchorage independent growth. BG-1 infected clones were tested for tumorigenicity by injection of cells (106 cells in 0.1cm2 50% matrigel; Collaborative Biomedical Products, Bedford, MA, USA) into subcutaneous sites in 6-week-old athymic Ncr-nude mice (NCI Animal Program, Bethesda, MD, USA) that were ovariectomized at approximately 4 weeks of age. Half of the ovariectomized mice received an implanted 0.18mg estrogen 60-day pellet (Innovative Research of America, Sarasota, FL, USA).","divisions":[{"label":"Title","span":{"begin":0,"end":22}}],"tracks":[]}