Figure 3

Doxycycline-mediated gene regulation in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats. (A-F) Doxycycline-controlled EGFP expression in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-luc rats was visualized by immunohistochemistry on sagittal sections using an antibody against EGFP. (A-C) In the absence of Dox, strong but mosaic EGFP expression is found in the cortex and hippocampus (HC). (D-F) In adult rats, EGFP expression could be completely inhibited by chronic Dox treatment (1 mg/mL drinking water) from conception until analyses. (G-I) Dual-label fluorescent immunohistochemistry of brain slices with the neuronal marker NeuN and EGFP. (I) Co-localization of EGFP and NeuN was frequently found in the CA1 region of the HC indicating Ptet-controlled reporter gene expression in the absence of Dox. (J) CaMKIIα-promoter controlled tTA activity was directly visualized by EGFP fluorescence on sagittal brain sections of double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats in the HC. Strong expression is mainly found in neurons of the CA1 and CA3 region. (K,L) Level of luciferase activity in different brain regions in the absence (-Dox, black) and presence of Dox (+Dox, 1 mg/mL, lightly shaded). The measured double transgenic animals were obtained by crossing the CaMKIIα-tTA line 4.5 to the EGFP-Ptetbi-luc 66.1 reporter line. (K) Animals were treated with Dox from conception throughout life until the day of analyses at the age of two months (+Dox, n = 5). Untreated control animals were measured at the same age (-Dox, n = 8). In Dox-treated CaMKIIα-tTA/EGFP-Ptetbi-luc rats, virtually no luciferase activity could be detected, while strong luciferase activity was found in all forebrain-specific regions (cortex, HC, olfactory bulb) of untreated rats (-Dox), leading to a highly significant difference between Dox-treated and untreated rats (***P ≤ 0.001). (L) To assess whether reporter gene expression could be suppressed with Dox in adult rats (+Dox adult), double transgenic animals, which had previously not received Dox, were treated with Dox for a period of three weeks during adulthood (-Dox, n = 10, +Dox adult, n = 6). Administration of Dox during adulthood suppressed luciferase activity, leading to a significant difference compared to untreated rats (***P ≤ 0.001). All data are presented as mean values + standard error of the mean. Logarithmic data transformation was performed prior to statistical analysis. Stars represent P-values obtained by unpaired t-test. ***P ≤ 0.001. Light units are normalized to the protein content of the lysates. Scale bar: 100 μm. Dox: doxycycline hydrochloride; EGFP: enhanced green fluorescent protein; HC: hippocampus.