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

The subcloning primers used in this study are summarized in Supplementary Table 1 Supplementary Table 1 and digested by arbitrary restriction enzymes, respectively. Lentiviral selfinactivating (SIN) vectors were kindly provided by Dr Miyoshi of the RIKEN BioResource Center (BRC), Ibaraki, Japan. CSII-CMV-MCS-IRES2-Venus plasmid coding murine IFN-b were constructed by inserting the ORF region digested by NheI and NotI from pcDNA3.1-IFN-b-venus. Experiments using plasmids inserted with genes coding for the fluorescence protein 'Venus', were performed by Dr Miyawaki of the RIKEN Brain Science Institute. To manufacture an insertion region for pUNO-interferon-stimulated response element (ISRE)-luc, the mammalian expression pUNO vector (Invivogen), which contains a strong and ubiquitous composite promoter designated EF1/HTLV, was used. The PCR products were amplified from pTAL-luc (Promega) with specific primers for a single-round PCR or with two-round PCR (Supplementary Table 1 ). The PCR products were inserted into the pCR-TOPO vector (Invitrogen) and digested by XhoI and NheI after they were confirmed by sequencing. Finally, the pUNO-ISRE-luc plasmid was constructed by inserting the purified product into the ORF region of the pUNO vector.