Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3

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

Generation of luciferase reporter plasmids. The information on primers used in this study is summarized in Table 1 . In order to analyze the 59-flanking region of the murine IRF-3 gene for promoter activity, PCR products amplified from mouse genomic DNA were subcloned into pGL3-Basic Vector (Promega) : pGL3 -2000/-1001 (nt 22000 to -1001, where the first nucleotide of IRF-3 exon 1 has been designated 1 1), pGL3 -1000/-524 (nt -1000 to -524), pGL3 -1000/-1 (nt -1000 to -1), pGL3 -523/-1 (nt -523 to -1), pGL3 -340/-1 (nt -340 to -1) and pGL3 -119/-1 (nt -119 to -1). Two different mutations (M1: ATTTGCAT to CGTTGCAT or M2: ATTTGCAT to GGGGAACC) were introduced into the Oct-1 site in the pGL3 -1000/-1, pGL3 -523/-1 and pGL3 -119/-1, generating pGL3 -1000/-1 (M1), pGL3 -523/-1 (M1), pGL3 -119/-1 (M1) and pGL3 -119/-1 (M2). pGL3 -119/-1 (M3: TTTCCCAC to GGGGGGGG) was the E2F transcription factor 1 (E2F1) sitemutated plasmid. pGL3 -119/-1 (M4: TGCGGT to TGCGCG or M5: TGCGGT to TCGGGT) were the acute myeloid leukemia 1 protein (AML1) site-mutated plasmids. Murine Oct-1 cDNA (NCBI Reference Sequence: NM_198932.2) was amplified by RT-PCR from total mRNA of N2a58 cells and subcloned into pcDNA3.1 (Invitrogen) to generate hemagglutinin (HA)-tagged Oct-1 expression plasmid (pcDNA Oct-1-HA). All generated plasmids were confirmed by sequencing.