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 the MSCV virus, RetroPack PT67 (Clontech) cells, containing the Moloney murine leukemia virus (MoMuLV) gag, pol, and env (10A1 virus-derived) genes, were transfected with either pMSCV-IFNAR1 or -Large T plasmids using Lipofectamine Õ 2000 (Invitrogen) after adding 25 mM chloroquine for 1 h prior to transfection. The medium was changed to a fresh growth medium 8 h after transfection. The culture supernatants were collected 72 h after medium replacement and filtrated with a 0.45 mm cellulose acetate membrane Nalgene Syringe Filter (Thermo). For measurement of MSCV virus titration, NIH3T3 cells were seeded at densities of 10 5 cells per well in 6-well plates and grown in a humidified incubator at 37 C and 5% CO 2 overnight to 70-80% confluence. The cells were treated with serial diluted viral solution (10 À1 to 10 À12 ) with 4 mg/ml polybrene and incubated for 24 h. The remaining colonies in each well were measured by Crystal violet staining after selection with antibiotics for 1 week. To prepare the lentivirus, HEK293T cells were co-transfected with these constructs and lentiviral packaging vectors (SIN vector plasmid: CSII-CMV-IRES2, packaging plasmid: pCAG-HIVgp and VSV-G/Rev plasmid: pCMV-VSV-G-RSV-Rev) using Lipofectamine Õ LTX (Invitrogen). After 16 h, the transfected cells were added to 10 mM forskolin. After 48 h, the growth medium, including the lentivirus, was collected and filtrated with 0.45 mm cellulose acetate membranes, and concentrated by the Lenti-X TM Concentrator as per the manufacturer's instructions (Clontech). The resultant lentivirus titration was checked by quantitating the p24 protein using the Lenti-X TM p24 Rapid Titer Kit (Clontech) in the culture medium.