Figure 4

Cloning-free construction and evaluation of de novo minimal promoters. (A, B) The reporter expression plasmid was used as a template for PCR. Forward primer contains at 3' end, 18 bases that target 5'UTR (exon 1 of the CMV IE gene) upstream of reporter cDNA and 5' end sequence representing 48 bases derived from CMV (control), RPS2, RPL39 promoters or the minimal inducible IFNB promoter (Table 1, SEQ 7–9). The reverse universal primer targets a region downstream of the polyA site. (C) The resultant and purified PCR products were transfected onto HEK293 cells. After 48 hrs, the GFP fluorescence was quantitated as outlined in the Figure 1 legend.(D) TLR-3 stably transfected HEK293 cells (TRL3+) or control (TLR3 negative) HEK293 cells were transiently transfected with IFNB minimal promoter-harboring PCR products and were treated with or without 25 μg/ml of poly I. C. Fluorescence levels were quantitated after 20 hr as described above.