The genomic sequence of the mouse TTP gene (GenBank accession number NT_187034) was used to engineer PCR cloning primers: 5′-GAGCTC TTCTATCTTTCTGTAACCCAC-3′ , 5′-CTCGAG TGGCAGAGAGATCCATGGTGG-3′ . Underlined letters are restriction enzyme sites. A 1309-base pair (bp) genomic fragment containing the 5′-flanking region of the TTP gene was isolated by PCR amplification from mouse genomic DNA. Construct pGL3/mTTP p-1309 contains the -1309 bp promoter region of the mouse TTP gene up to nucleotide +48 base pair (i.e., downstream from the mouse TTP mRNA cap site) inserted into the SacI and XhoI sites of the pGL3 basic vector (Promega). A pRL-SV40 Renilla luciferase construct was purchased from Promega (E2231).
RAW264.7 cells were transfected with luciferase reporter constructs using the Neon™ transfection system (Invitrogen). Lysates of the transfected cells were mixed with luciferase assay reagent (Promega), and the chemiluminescent signal was measured in a Wallac Victor 1420 Multilabel Counter (EG&G Wallac, Turku, Finland). Firefly luciferase of pGL3/mTTP p-1309 was normalized to Renilla luciferase of pRL-SV40 in each sample.
RAW264.7 cells were transfected with luciferase reporter constructs using the Neon™ transfection system (Invitrogen). Lysates of the transfected cells were mixed with luciferase assay reagent (Promega), and the chemiluminescent signal was measured in a Wallac Victor 1420 Multilabel Counter (EG&G Wallac, Turku, Finland). Firefly luciferase of pGL3/mTTP p-1309 was normalized to Renilla luciferase of pRL-SV40 in each sample.