Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

To prepare primary mouse embryonic fibroblasts (MEFs), mouse embryos from the C57BL/6 and Ifnar1 À/À pregnant female mice were isolated and dissected out of the uterine horns at Days 13 to 15. After rinsing them in 70% ethanol, each embryo was separated from its placenta, and the brain and internal organs were removed and placed in a petri dish containing phosphate-buffered saline (PBS). Sequentially, the tissues were chopped using razor blades and were suspended and subdivided by trypsin-EDTA. All of these procedures were performed using aseptic technique in a biological safety cabinet. After centrifugation, these collected cells were resuspended and seeded with fresh DMEM, containing 10% FBS, as described above, in culture dishes. The medium was changed on the following day and the adhered fibroblast cells were cultured and passaged until adequate cell volume was achieved. To establish the immortalizing MEFs, the amplified fibroblasts from Ifnar1 À/À mice were subcloned and passaged by serial replacing with 50 to 100 mg/ml HygroGold TM selections of the cells, following infection of MSCV-Large T vector at a rate to 10 12 colony forming units (cfu) per 100 mm culture dish. The Ifnar1 gene-introduced Ifnar1 À/À MEFs were established by subcloning and passaging with 2.5 mg/ml puromycin. Selection of the cells occurred following infection with the MSCV-IFNAR1 vector with final concentration of 4 mg/ml polybrene at a rate to 10 12 cfu per 100 mm culture dish.