PMC:5608921 / 24656-26329 JSONTXT

{"project":"2_test","denotations":[{"id":"28846079-11063126-79081959","span":{"begin":378,"end":380},"obj":"11063126"}],"text":"Animal handling and treatments\nThe genetic background of Tg(K6b-E6/E7)-M8 transgenic mice (always hemizygous for the transgene) was that of CD1 outbred or Fvb/N inbred strains. These and the corresponding WT mice were maintained and treated in accordance with the regulations of the local Bioethical Committee. Genotype was determined by E7-specific PCR, as previously reported.26 Bilateral ovariectomy was performed at the moment of ear perforation, whereas castration in males was performed 2 weeks before ear punching. Pellets embedded with 0.05 mg ß-estradiol (E2) 17-acetate (90-day release; NE-271; Innovative Research of America, Sarasota, FL, USA) were implanted in the dorsal-anterior area of mice at the day of ear perforation for long-term ear regeneration experiments; whereas, pellets embedded with 0.25 mg E2 17-acetate (90-day release; NE-271) were implanted for estrous cycle determination (19 days) or for cancer experiments (up to 3 pellets for 9 months). For short-term regeneration studies, E2 (0.7 μg/100 μl dissolved in corn oil) or the vehicle were injected subcutaneously every day up to for 14 days; hormone was not injected on the day of ear perforation or of BrdU injection. For ER inhibition, female mice were intraperitoneally injected with raloxifene (Eli Lilly, Indianapolis, IN, USA; 10 mg/ml in phosphate-buffered saline (PBS)); for regeneration experiments, 1.5 mg of raloxifene was delivered daily for 14 days or 5 days/week for a month and, during the estrous cycle, the same amount was delivered daily through 19 days or once at proestrus. BrdU (50 μg/g weight of mice) was intraperitoneally injected (1 dose) and mice killed 2 h later."}