Isolation of the cirp genomic fragments that increase expression at moderately low temperatures. (A) Comparison of the degradation rates of reporter mRNA at 37°C and 32°C. NIH/3T3 Flp-In cells stably transfected with the pcDNA5/FRT vector expressing firefly luciferase (F-Luc) under the control of the cirp −970/+56 genomic region instead of the CMV promoter were transferred to 32°C or remained at 37°C for indicated times after starting incubation with 15 μg/ml actinomycin D (ActD). Expression of F-Luc was analyzed by northern blotting (20 μg of total RNA per lane), and compared with that of ribosomal protein S26 (left). Expression of F-Luc mRNA was quantified by quantitative RT-PCR, normalized to that of Tubb3 having a long mRNA half-life , and expressed as relative to time 0 (right). Values are mean ± SD, n = 3. Values do not significantly differ at 37°C and 32°C. (B) HEK293 cells were transiently co-transfected with CAT reporter constructs having the gene fragment upstream of the cirp transcription initiation site (+1) as indicated and plasmids expressing β-galactosidase (LacZ). The reporter constructs contained endogenous cirp promoter (upper panel) or SV40 minimal promoter (lower panel). Cells were maintained at 37°C or transferred to 32°C one day after transfection, and 36 hours later cell extracts were assayed. CAT protein level was normalized to LacZ activity. Bars represent the means ± SE of three to four determinations. a.u., arbitrary unit. *, Normalized CAT level at 32°C divided by that at 37°C. N/A, not applicable.