Location of the iron-responsive element in vivo. Mice receiving an iron-deficient diet (2-5 ppm) were hydrodynamically transfected with a pGL3 reporter plasmid containing the firefly luciferase gene (luc) under the control of various lengths of the murine hepcidin 1 (Hamp1) promoter. After 3 days, the basal level of bioluminescence was determined and mice were divided into 2 groups; 1 received a high-iron diet (2 × 10⁴ ppm), and the second group remained on the iron-deficient diet (2–5 ppm). After 24 hours, the mice were anesthetized and reinjected with luciferin, and the bioluminescence was remeasured (day 4). The day-4 bioluminescence is expressed as fold change over baseline day 3 bioluminescence. Because the reporter is delivered by hydrodynamic transfection that results in transient expression, the actual levels of expressed reporter decreased with time. As a result, without stimulation, the day-4 bioluminescence is about one-third of the day-3 bioluminescence. Thus, the fold change of day-4 bioluminescence over day-3 baseline bioluminescence in mice on an iron-deficient diet is less than 1. The number of base pairs upstream of the start of translation is given for each promoter construct. The construct designated 260 bp + (1.6 to 1.8 Kb) contains the first 260 bp and the portion of the promoter between 1.6 and 1.8 Kb after the start of translation. The number of animals in each group on iron-deficient and high-iron diets was equivalent and is shown in the brackets; error bars represent 1 SEM.