Generation of 4xhre-tata Hif-reporter lines. (A) Schematic representation of the Tol-2 vector used to generate the Tg(4xhre-tata:eGFP)ia21 line. The construct consists of a 98-bp fragment encoding four HREs (in red) from the murine lactate dehydrogenase followed by a TATA minimal promoter (in boldface), EGFP (in green), and the poly(A) signal. (B–D) Representative image of Tg(4xhre-tata:eGFP)ia21 embryos at 48 hpf injected with a control MO (B) or the HIFβ-MO MO (C). (Magnification: 5×.) Down-regulation of Hifβ significantly decreases transgene activity as reported by the integrated density analysis of fluorescence (D). (E–G) HIf-1α dominant active (DA) mRNAs injected in Tg(4xhre-tata:eGFP)ia21 embryos increase transgene expression (F) compared with control embryos (E). (Magnification: 5×.) (H–J) Treatment from 72 hpf to 96 hpf with 100 μM DMOG significantly increases 4xhre-tata transgene activity (I) with respect to the control embryos (H). (Magnification: 4×.) (D, G, and J) Average values of fluorescence integrated density calculated for treated embryos and controls. Values represent the mean ± SEM (***P < 0.001; **P < 0.01; *P < 0.05). A.U., arbitrary units.
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